WO2020037258A1 - Anticorps catalytiques et leurs méthodes d'utilisation - Google Patents

Anticorps catalytiques et leurs méthodes d'utilisation Download PDF

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WO2020037258A1
WO2020037258A1 PCT/US2019/046903 US2019046903W WO2020037258A1 WO 2020037258 A1 WO2020037258 A1 WO 2020037258A1 US 2019046903 W US2019046903 W US 2019046903W WO 2020037258 A1 WO2020037258 A1 WO 2020037258A1
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amino acid
seq
acid sequence
catabody
level
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PCT/US2019/046903
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English (en)
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Yue Liu
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Ab Studio Inc.
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Priority to EP19849955.0A priority Critical patent/EP3836966A4/fr
Priority to US17/268,933 priority patent/US20210190796A1/en
Priority to CN201980054508.0A priority patent/CN112584863A/zh
Publication of WO2020037258A1 publication Critical patent/WO2020037258A1/fr
Priority to US18/045,114 priority patent/US20230258656A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0002—Antibodies with enzymatic activity, e.g. abzymes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/564—Immunoassay; Biospecific binding assay; Materials therefor for pre-existing immune complex or autoimmune disease, i.e. systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, rheumatoid factors or complement components C1-C9
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6854—Immunoglobulins
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G01N33/6896—Neurological disorders, e.g. Alzheimer's disease
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/567—Framework region [FR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/901—Antibodies with enzymatic activity; e.g. abzymes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/28—Neurological disorders
    • G01N2800/2814—Dementia; Cognitive disorders
    • G01N2800/2821—Alzheimer

Definitions

  • the present application is related to catabodies, methods for determining catabody levels, and methods of treatment using catabodies.
  • the present application is related to compositions and use of catabodies specifically recognizing and cleaving amyloid beta (Ab) peptides.
  • Catabodies are antibodies that specifically bind to target antigens and catalyze chemical transformation of their target antigens.
  • Proteolytic catabodies can hydrolyze and permanently inactivate target peptides.
  • a single catabody molecule can hydrolyze thousands of antigen molecules over its biological lifetime, thereby achieving enhanced potency compared to a stoichiometrically-binding conventional antibody.
  • Naturally-occurring catabodies have been found in normal humans and in patients with autoimmune diseases.
  • Catabodies have also been raised in lab animals immunized against synthetic haptens or screened from antibody libraries using transition-state analogs. However, due to their relatively modest catalytic activity, catabodies have not been widely developed as therapeutic agents.
  • Protein aggregation is a biological phenomenon in which misfolded proteins aggregate intracellularly or extracellularly. These protein aggregates are often associated with neurodegenerative diseases including Amyotrophic lateral sclerosis (ALS), Alzheimer’s disease (AD), Parkinson's disease (PD), prion diseases and other amyloidosis diseases.
  • ALS Amyotrophic lateral sclerosis
  • AD Alzheimer’s disease
  • PD Parkinson's disease
  • prion diseases other amyloidosis diseases.
  • soluble and fibrillary amyloid beta (Ab) peptides aggregates contribute causally to the pathogenesis of AD.
  • the Ab aggregates activate microglial inflammatory processes, exert direct neurotoxic effects, and disrupt the anatomic architecture of the brain.
  • antibody-mediated antibody include antigen binding
  • compositions and methods for diagnosis including methods of determining a risk
  • treatment and prevention of a protein aggregation disease (PAD) such as Alzheimer’s disease (AD) in an individual.
  • PAD protein aggregation disease
  • AD Alzheimer’s disease
  • One aspect of the present application provides a method for determining the level of one or more SHD catabodies (i.e., catabodies containing a catalytic triad motif of serine, histidine and aspartate, or“SHD motif’) in a biological sample, comprising: a) contacting the biological sample with a substrate peptide immobilized on a solid support under conditions that allow formation of a catabody-substrate peptide complex, and b) determining the amount of the catabody-substrate peptide complex, thereby providing the level of one or more SHD catabodies in the biological sample, wherein the substrate peptide comprises the amino acid sequence (EAR) n (SEQ ID NO: 2), and wherein n is an integer between 1 and 30 (e.g.
  • the biological sample is incubated with the substrate peptide for about 1 hour to about 16 hours, such as any one of about 1 hour to about 3 hours, about 3 hours to about 8 hours, or about 8 hours to about 16 hours.
  • the biological sample is a serum sample.
  • the serum sample contains at least about 1 mg/mL (e.g., at least about 10 mg/mL, 25 mg/mL, or 100 mg/mL)
  • immunoglobulin (Ig; e.g., human Ig).
  • the amount of the catabody-substrate peptide complex is determined using an antibody that specifically binds to total immunoglobulin (Ig, such as total human Ig).
  • Ig total immunoglobulin
  • the antibody specifically binds to total IgM, total lgG, total IgA, and/or total IgE.
  • the antibody is labeled with an enzyme (e.g., horseradish peroxidase or“HRP”) or a fluorescent label (e.g., FITC).
  • HRP horseradish peroxidase
  • FITC fluorescent label
  • One aspect of the present application provides a method for determining a risk for a PAD in an individual, wherein the PAD is associated with aggregation of a target protein, comprising determining the level of one or more SHD catabodies in a biological sample (e g., serum sample) of the individual, wherein the individual is determined as having a risk for the PAD if the level of the one or more SHD catabodies is lower than a control SHD catabody level.
  • the level of one or more SHD catabodies is the level of one or more SHD catabodies that specifically bind to the target protein.
  • the level of one or more SHD catabodies is the level of total SHD catabodies.
  • the level of total SHD catabodies is determined by contacting a serum sample of the individual with a substrate peptide immobilized on a solid support under conditions that allow formation of a catabody-substrate peptide complex, and determining the amount of the catabody-substrate peptide complex, wherein the substrate peptide comprises the amino acid sequence (EAR) n (SEQ ID NO: 2), and wherein n is an integer between 1 and 30 (e.g., n is 3).
  • the serum sample contains at least about 1 mg/mL (e.g., at least about 10 mg/mL, 25 mg/mL, or 100 mg/mL) Ig (e.g, human Ig).
  • the biological sample is incubated with the substrate peptide for about 1 hour to about 16 hours, such as any one of about 1 hour to about 3 hours, about 3 hours to about 8 hours, or about 8 hours to about 16 hours.
  • the amount of the catabody-substrate peptide complex is determined using an antibody that specifically binds to total Ig (e.g, total human Ig).
  • the antibody specifically binds to total IgM, total IgG, total IgA, and/or total IgE.
  • the antibody is labeled with an enzyme (e.g, horseradish peroxidase or“HRP”) or a fluorescent label (e.g, FTTC).
  • the method further comprises determining the level of an auto-antibody against the target protein in a biological sample (e.g, serum sample) of the individual, and wherein the individual is determined as having a risk for the PAD if: (i) the level of the one or more SHD catabodies is lower than a control SHD catabody level; and (ii) the level of the auto-antibody against the target protein is lower than a control auto-antibody level.
  • a biological sample e.g, serum sample
  • the level of the auto-antibody is determined by contacting a serum sample of the individual with the target protein under conditions that allow formation of an auto- antibody-target protein complex, and determining the amount of the auto-antibody-target protein complex. In some embodiments, the level of the auto-antibody is determined using an ELISA assay. In some embodiments, the control auto-antibody level is the level of the auto- antibody against the target protein in a healthy individual (e.g. , of the same age group). In some embodiments, the control auto-antibody level is the median level of the auto-antibody against the target protein in a population of individuals (e.g. , of the same age group).
  • the method further comprises determining the level of the target protein in a biological sample (e.g, serum sample or cerebrospinal fluid sample) of the individual, and wherein the individual is determined as having a risk for the PAD if: (i) the level of the one or more SHD catabodies is lower than a control SHD catabody level; and (ii) the level of the target protein is higher than a control target protein level.
  • the level of the target protein is determined by contacting the biological sample of the individual with an antibody against the target protein under conditions that allow formation of an antibody- target protein complex, and determining the amount of the antibody-target protein complex.
  • the level of the target protein is determined using an ELISA assay.
  • the control target protein level is the level of the target protein in a healthy individual (e.g, of the same age group). In some embodiments, the control target protein level is the median level of the target protein in a population of individuals (e.g, of the same age group).
  • control SHD catabody level is the level of one or more SHD catabodies in a healthy individual (e.g, of the same age group). In some embodiments, the control SHD catabody level is the median level of one or more SHD catabodies in a population of individuals (e.g, of the same age group).
  • the PAD is Alzheimer’s disease, and wherein the target protein is amyloid b (Ab).
  • the PAD is Parkinson’s disease, and the target protein is a- synuclein.
  • the PAD is Alzheimer’s disease or dementia, and the target protein is Tau.
  • the PAD is ATTR amyloidosis, and the target protein is transthyretin.
  • the PAD is AL amyloidosis, and the target protein is immunoglobulin light chain.
  • the PAD is frontotemporal lobar degeneration (FTLD) or amyotrophic lateral sclerosis (ALS), and the target protein is TDP43.
  • FTLD frontotemporal lobar degeneration
  • ALS amyotrophic lateral sclerosis
  • the target protein is TDP43.
  • the PAD is Huntington’s disease, and the target protein is Huntingtin.
  • the PAD is Type P diabetes, and the target protein is LAPP.
  • the PAD is Amyotrophic Lateral Sclerosis (ALS), and the target protein is SOD1.
  • One aspect of the present application provides a method of treating or preventing a protein aggregation disease (PAD) in an individual, wherein the PAD is associated with aggregation of a target protein, comprising: a) determining the individual as having a risk for the PAD according to any one of the methods for determining a risk as described above; and b) administering to the individual an effective amount of a therapeutic catabody that specifically binds to the target protein.
  • the method is repeated at a frequency of no mote than about every three months, e.g., about every three months, about every six months, or about every year.
  • the PAD is Alzheimer’s disease and wherein the target protein is amyloid b (Ab), and wherein the therapeutic catabody comprises a light chain variable region (VL) comprising a light chain complementarity determining region (LC-CDR) 1 comprising the amino acid sequence of SEQ ID NO: 12, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 13, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 14, or a variant thereof comprising up to about 5 (e.g., 1, 2, 3, 4, or 5) amino acid substitutions in the LC-CDRs, wherein the amino acid residue at position 1 of the VL is D, the amino acid residue at position 27A of the VL is S, and the amino acid residue at position 93 of the VL is H, and wherein the numbering is according to the EU index of Rabat.
  • VL light chain variable region
  • LC-CDR light chain complementarity determining region
  • the therapeutic catabody comprises a heavy chain variable region (VH) comprising a heavy chain complementarity determining region (HC-CDR) 1 comprising the amino acid sequence of SEQ ID NO: 9, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 10, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 11, or a variant thereof comprising up to about 5 (e.g. , 1, 2, 3, 4, or 5) amino acid substitutions in the HC-CDRs.
  • VH heavy chain variable region
  • HC-CDR heavy chain complementarity determining region
  • the amino acid residue at position 26 of the VL is S
  • the amino acid residue at position 27D of the VL is D, E or H
  • the amino acid residue at position 28 of the VL is D or N
  • the numbering is according to the EU index of Rabat.
  • the therapeutic catabody comprises a VH comprising an amino acid sequence having at least about 85% sequence identity (e.g., at least about any one of 90%, 95%, 97%, 99%, or 100%) to the amino acid sequence of SEQ ID NO: 4, 6, 19 or 20.
  • the therapeutic catabody comprises a VL comprising an amino acid sequence having at least about 85% (e.g, at least about any one of 90%, 95%, 97%, 99%, or 100%) sequence identity to the amino acid sequence of SEQ ID NO: 5, 7, 8, 21 or 22.
  • the therapeutic catabody comprises a VH comprising an amino acid sequence selected from the amino acid sequences of SEQ ID NOs: 4, 6, 19 and 20 and a VL comprising an amino acid sequence selected from the amino acid sequences of SEQ ID NO: 5, 7, 8, 21 and 22.
  • the therapeutic catabody comprises: (i) a VH comprising the amino acid sequence of SEQ ID NO: 4, and a VL comprising the amino acid sequence of SEQ ID NO: 5; (ii) a VH comprising the amino acid sequence of SEQ ID NO: 6, and a VL comprising the amino acid sequence of SEQ ID NO: 7; (iii) a VH comprising the amino acid sequence of SEQ ID NO: 6, and a VL comprising the amino acid sequence of SEQ ID NO: 8; (iv) a VH comprising the amino acid sequence of SEQ ID NO: 19, and a VL comprising the amino acid sequence of SEQ ID NO: 21 ; (v) a VH comprising the amino acid sequence of SEQ ID NO: 20, and a VL comprising the amino acid sequence of SEQ ID NO: 21; (vi) a VH comprising the amino acid sequence of SEQ ID NO: 19, and a VL comprising the amino acid sequence of SEQ ID NO: 22; or (vii)
  • the therapeutic catabody is a full-length IgG antibody. In some embodiments, the therapeutic catabody comprises an IgGl or IgG4 Fc region. In some embodiments, the therapeutic catabody is a full-length IgM antibody.
  • Another aspect of the present application provides an isolated anti-Ab catabody comprising: a VL comprising a LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 12, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 13, and an LC- CDR3 comprising the amino acid sequence of SEQ ID NO: 14, or a variant thereof comprising up to about 5 (e.g., 1, 2, 3, 4, or 5) amino acid substitutions in the LC-CDRs, wherein the amino acid residue at position 1 of the VL is D, the amino acid residue at position 27A of the VL is S, and the amino acid residue at position 93 of the VL is H, and wherein the numbering is according to the EU index of Kabat.
  • the anti-Ab catabody comprises VH comprising an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 9, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 10, and an HC-CDR3 comprising the amino acid sequence of SEQ P) NO: 11, or a variant thereof comprising up to about 5 (e.g. , 1 , 2, 3, 4, or 5) amino acid substitutions in the HC-CDRs.
  • the isolated anti-Ab catabody comprises: a VH comprising an HC-CDR1 comprising the amino acid sequence of SEQ 1D NO: 9, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 10, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 11; and a VL comprising an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 12, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 13, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 14.
  • an isolated anti-Ab catabody comprising: a VH comprising an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 9, an HC- CDR2 comprising the amino acid sequence of SEQ ID NO: 10, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 11, or a variant thereof comprising up to about 5 (e.g., 1, 2, 3, 4, or 5) amino acid substitutions in the HC-CDRs, wherein the amino acid residue at position 1 of the VL is D, the amino acid residue at position 27A of the VL is S, and the amino acid residue at position 93 of the VL is H, and wherein the numbering is according to the EU index of Rabat.
  • the isolated anti-Ab catabody comprises: a VL comprising a LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 12, an LC- CDR2 comprising the amino acid sequence of SEQ ID NO: 13, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 14, or a variant thereof comprising up to about 5 (e.g., 1, 2, 3, 4, or 5) amino acid substitutions in the LC-CDRs.
  • the isolated anti-Ab catabody comprises: a VH comprising an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 9, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 10, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 11 ; and a VL comprising an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 12, an LC- CDR2 comprising the amino acid sequence of SEQ ID NO: 13, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 14.
  • an isolated anti-Ab catabody comprising: a VH comprising an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 9, an HC- CDR2 comprising the amino acid sequence of SEQ ID NO: 10, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 11; and a VL comprising an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 12, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 13, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 14, wherein the amino acid residue at position 1 of the VL is D, the amino acid residue at position 27A of the VL is S, and the amino acid residue at position 93 of the VL is H, and wherein the numbering is according to the EU index of Rabat.
  • the anti-Ab catabody cleaves a substrate having the formula EAR-AMC (SEQ ID NO:
  • the amino acid residue at position 26 of the VL is S
  • the amino acid residue at position 27D of the VL is D, E or H
  • the amino acid residue at position 28 of the VL is D or N
  • the numbering is according to the EU index of Kabat.
  • the anti-Ab catabody comprises a VH comprising an amino acid sequence having at least about 85% sequence identity (e.g., at least about any one of 90%, 95%, 97%, 99%, or 100%) to the amino acid sequence of SEQ ID NO: 4, 6, 19 or 20.
  • the anti-Ab catabody comprises a VL comprising an amino acid sequence having at least about 85% (e.g., at least about any one of 90%, 95%, 97%, 99%, or 100%) sequence identity to the amino acid sequence of SEQ ID NO: 5, 7, 8, 21 or 22.
  • the anti-Ab catabody comprises a VH comprising an amino acid sequence selected from the amino acid sequences of SEQ ID NOs: 4, 6, 19 and 20 and a VL comprising an amino acid sequence selected from the amino acid sequences of SEQ ID NO: 5, 7, 8, 21 and 22.
  • the anti-Ab catabody comprises: (i) a VH comprising the amino acid sequence of SEQ P) NO: 4, and a VL comprising the amino acid sequence of SEQ ID NO: 5; (ii) a VH comprising the amino acid sequence of SEQ ID NO: 6, and a VL comprising the amino acid sequence of SEQ ID NO: 7; (iii) a VH comprising the amino acid sequence of SEQ ID NO: 6, and a VL comprising the amino acid sequence of SEQ ID NO: 8; (iv) a VH comprising the amino acid sequence of SEQ ID NO: 19, and a VL comprising the amino acid sequence of SEQ ID NO: 21 ; (v) a VH comprising the amino acid sequence of SEQ ID NO: 20, and a VL comprising the amino acid sequence of SEQ ID NO: 21; (vi) a VH comprising the amino acid sequence of SEQ ID NO: 19, and a VL comprising the amino acid sequence of SEQ ID NO: 22; or
  • the anti-Ab catabody is a full-length IgG antibody.
  • the anti-Ab catabody comprises an IgGl or IgG4 Fc region.
  • the anti-Ab catabody is a full-length IgM antibody.
  • Alzheimer’s disease in an individual comprising administering to the individual an effective amount of an anti-Ab catabody according to any one of the anti-Ab catabodies described above.
  • an anti-Ab catabody according to any one of the anti-Ab catabodies described above.
  • kits for treating or preventing Alzheimer’s disease in an individual comprising: a) a substrate peptide comprises the amino acid sequence (EAR) n (SEQ ID NO: 2), and wherein n is an integer between 1 and 30 (e.g., n is 3); b) an Ab peptide; and c) an antibody that specifically binds to total Ig (e.g., total human Ig).
  • the antibody specifically binds to total IgM, total IgG, total IgA, and/or total IgE.
  • the kit further comprises a solid support, such as an ELISA plate.
  • the kit further comprises a therapeutic catabody that specifically binds to Ab.
  • FIG. 1A shows catabody levels in the serum samples of young (20-29 years old) and old (60-69 years old) adults as determined by an EAR-AMC binding assay.
  • FIG. IB shows catabody levels in the serum samples of young (20-29 years old) and old (60-69 years old) adults as determined by an EARS binding assay.
  • FIG. 2 shows catabody and Ab-specific auto-antibody levels in healthy (HS1-HS8) individuals or patients with Alzheimer’s disease (ALZ-1-5).
  • FIG. 3 shows alignment of VL sequences of two catabodies (anti-UA15 and anti-VP) and non-catalytic anti-Ab antibody 3D6. Amino acid residues corresponding to the SHD motif are marked with asterisk (*). Amino acid residues that may support catalytic function of the catabodies are marked with pound (#).
  • FIG. 4 shows an SDS gel image of various purified 3D6-derived catabodies under non-reducing and reducing electrophoresis conditions.
  • 3D6-D is a catabody designed based on 3D6 (i.e., 3D6-Y).
  • hu3D6-D H1L1 and hu3D6-D H1L2 are humanized versions of 3D6-D.
  • hu3D6-Y has the same sequences as bapineuzumab (i.e., humanized 3D6).
  • FIG. 5 shows catalytic function of 3D6-D as determined using an EAR-AMC substrate.
  • FIG. 6 shows binding of 3D6-D to (EAR) 3 (SEQ ID NO: 3) and Ab.
  • FIG. 7 shows binding of humanized 3D6 catabodies with Ab.
  • FIG. 8 shows anti-Ab autoantibody levels and SHD catabody (recognizing (EAR) 3 ) levels in the serum of 30 Alzheimer’s disease (AD) patients measured by ELISA assay.
  • PBS served as negative control.
  • the average readout from pooled healthy human serum and serum samples of 8 healthy donors served as control.
  • The“% of Control” was calculated as (readout of AD sample) divided by (average readout of pooled human semm and serum of 8 healthy donors).“+” in the table indicates the semm level of anti-Ab autoantibody or SHD catabody in AD patient semm is higher than that of healthy donor and pooled serum samples.
  • serum level of anti-Ab autoantibody or SHD catabody in AD patient serum is lower than that of healthy donor and pooled semm samples.
  • FIG. 9A depicts ELISA experimental design for detecting anti-Ab autoantibody levels and SHD catabody (recognizing (EAR)a) levels in the serum of 30 Alzheimer’s disease (AD) patients, 8 healthy donors, and pooled healthy donor semm sample. PBS served as negative control.
  • FIG. 9B depicts plate reading for Ab ELISA binding assay at 5 min time point.
  • FIG. 9C depicts plate reading for (EAR)? ELISA binding assay at 1 min time point.
  • the present application in one aspect provides methods for diagnosis and treatment of a protein aggregation disease (PAD) using a newly developed immunoassay for determining SHD catabody levels in a biological sample.
  • PAD protein aggregation disease
  • the present application is based in part on the discover)' that SHD catabody levels in the semm correlate with auto-antibody levels against amyloid beta (Ab) peptides in aging individuals and patients having Alzheimer’s disease (AD).
  • AD amyloid beta
  • imaging is still the most reliable diagnosis method for AD.
  • the disease shows clear pathologic features in the imaging analysis, neural damage has already occurred. At this stage, even if a therapeutic agent can prevent further progression of the disease, the therapeutic agent is unlikely to reverse the neural damage.
  • the present invention allows effective diagnosis and treatment of PAD (such as AD) at an early stage using SHD catabody levels as an early diagnostic biomarker.
  • an individual having a risk of AD can be determined at an early stage using the methods described herein, and treated with an anti-Ab catabody described herein to prevent AD.
  • one aspect of the present application provides a method for determining a risk for a PAD in an individual, wherein the PAD is associated with aggregation of a target protein, comprising determining the level of one or more SHD catabodies in a biological sample (e.g, serum sample) of the individual, wherein the individual is determined as having a risk for the PAD if the level of the one or more SHD catabodies is lower than a control SHD catabody level.
  • a biological sample e.g, serum sample
  • the method comprises: a) contacting a serum sample of the individual with a substrate peptide immobilized on a solid support under conditions that allow formation of a catabody-substrate peptide complex, and b) determining the amount of the catabody-substrate peptide complex, thereby providing the level of one or more SHD catabodies in the biological sample, wherein the substrate peptide comprises the amino acid sequence (EAR)n (SEQ ID NO: 2), and wherein n is an integer between 1 and 30.
  • EAR amino acid sequence
  • the method further comprises determining the level of an autoantibody against the target protein in a biological sample (e.g., serum sample) of the individual, and wherein the individual is determined as having a risk for the PAD if: (i) the level of the one or more SHD catabodies is lower than a control SHD catabody level; and (ii) the level of the auto-antibody against the target protein is lower than a control auto-antibody level.
  • a biological sample e.g., serum sample
  • One aspect of the present application provides a method of treating or preventing a PAD (e.g, AD) in an individual, wherein the PAD is associated with aggregation of a target protein (e.g, Ab), comprising: a) determining the level of one or more SHD catabodies in a biological sample (e.g, serum sample) of the individual, wherein the individual is determined as having a risk for the PAD if the level of the one or more SHD catabodies is lower than a control SHD catabody level; and b) administering to the individual an effective amount of a therapeutic catabody that specifically binds to the target protein (e.g, Ab).
  • a biological sample e.g, serum sample
  • step a) comprises: 1) contacting a serum sample of the individual with a substrate peptide immobilized on a solid support under conditions that allow formation of a catabody-substrate peptide complex, and 2) determining the amount of the catabody-substrate peptide complex, thereby providing the level of one or more SHD catabodies in the serum, wherein the substrate peptide comprises the amino acid sequence (EAR)n (SEQ ID NO: 2), and wherein n is an integer between 1 and 30.
  • EAR amino acid sequence
  • step a) further comprises determining the level of an auto-antibody against the target protein in a biological sample (e.g., serum sample) of the individual, and wherein the individual is determined as having a risk for the PAD if: (i) the level of the one or more SHD catabodies is lower than a control SHD catabody level; and (ii) the level of the auto-antibody against the target protein is lower than a control auto-antibody level.
  • a biological sample e.g., serum sample
  • Another aspect of the present application provides an isolated anti-Ab catabody comprising a light chain variable region (VL) comprising a light chain complementarity determining region (LC-CDR) 1 comprising the amino acid sequence of SEQ ID NO: 12, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 13, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 14, or a variant thereof comprising up to about 5 amino acid substitutions in the LC-CDRs, wherein the amino acid residue at position 1 of the VL is D, the amino acid residue at position 27A of the VL is S, and the amino acid residue at position 93 of the VL is H, and wherein the numbering is according to the EU index of Kabat.
  • VL light chain variable region
  • LC-CDR light chain complementarity determining region
  • the anti-Ab catabody comprises a heavy chain variable region (VH) comprising a heavy chain complementarity determining region (HC-CDR) 1 comprising the amino acid sequence of SEQ 1D NO: 9, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 10, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 11 , or a variant thereof comprising up to about 5 amino acid substitutions in the HC-CDRs.
  • VH heavy chain variable region
  • HC-CDR heavy chain complementarity determining region
  • compositions such as pharmaceutical compositions
  • kits and articles of manufacture for diagnosis, treatment or prevention of PAD e.g, AD
  • beneficial or desired clinical results include, but are not limited to, one or more of the following: alleviating one or more symptoms resulting from the disease, diminishing the extent of the disease, stabilizing the disease (e.g, preventing or delaying the worsening of the disease), preventing or delaying the spread of the disease, preventing or delaying the recurrence of the disease, delay or slowing the progression of the disease, ameliorating the disease state, providing a remission (partial or total) of the disease, decreasing the dose of one or more other medications required to treat the disease, delaying the progression of the disease, increasing or improving the quality of life, increasing weight gain, and/or prolonging survival.
  • the methods of the invention contemplate any one or more of these aspects of treatment.
  • the term“antibody” includes full-length antibodies and antigen-binding fragments thereof.
  • a full-length antibody comprises two heavy chains and two light chains.
  • the variable regions of the light and heavy chains are responsible for antigen binding.
  • the variable regions in both chains generally contain three highly variable loops called the complementarity determining regions (CDRs) (light chain (LC) CDRs including LC-CDR1, LC-CDR2, and LC-CDR3, heavy chain (HC) CDRs including HC-CDR1, HC-CDR2, and HC-CDR3).
  • CDRs complementarity determining regions
  • CDR boundaries for the antibodies and antigen-binding fragments disclosed herein may be defined or identified by the conventions of Rabat, Chothia, or Al-Lazikani (Al- Lazikani 1997; Chothia 1985; Chothia 1987; Chothia 1989; Rabat 1987; Rabat 1991).
  • the three CDRs of the heavy or light chains are interposed between flanking stretches known as framework regions (FRs), which are more highly conserved than the CDRs and form a scaffold to support the hypervariable loops.
  • FRs framework regions
  • the constant regions of the heavy and light chains are not involved in antigen binding, but exhibit various effector functions.
  • Antibodies are assigned to classes based on the amino acid sequence of the constant region of their heavy chain.
  • the five major classes or isotypes of antibodies are IgA, IgD, IgE, IgG, and IgM, which are characterized by the presence of a, 5, e, g, and m heavy chains, respectively.
  • IgGl g ⁇ heavy chain
  • IgG2 g2 heavy chain
  • IgG3 g3 heavy chain
  • IgG4 g4 heavy chain
  • IgAl al heavy- chain
  • IgA2 ct2 heavy chain
  • antigen-binding fragment refers to an antibody fragment including, for example, a diabody, a Fab, a Fab’, a F(ab’)2, an Fv fragment, a disulfide stabilized Fv fragment (dsFv), a (dsFv)2, a bispecific dsFv (dsFv-dsFv’), a disulfide stabilized diabody (ds diabody), a single-chain Fv (scFv), an scFv dimer (bivalent diabody), a multispecific antibody formed from a portion of an antibody comprising one or more CDRs, or any other antibody fragment that binds to an antigen but does not comprise a complete antibody structure.
  • an antigen-binding fragment is capable of binding to the same antigen to which the parent antibody or a parent antibody fragment (e.g., a parent scFv) binds.
  • an antigen-binding fragment may comprise one or more CDRs from a particular human antibody grafted to a framework region from one or more different human antibodies.
  • epitope refers to the specific group of atoms or amino acids on an antigen to which an antibody binds. Two antibodies may bind the same epitope within an antigen if they exhibit competitive binding for the antigen.
  • the term“specifically binds,”“specifically recognizing,” or“is specific for” refers to measurable and reproducible interactions, such as binding between a target and an antibody ( e.g . , catabody), that is determinative of the presence of the target in the presence of a heterogeneous population of molecules, including biological molecules.
  • an antibody that specifically recognizes a target is an antibody that binds this target with greater affinity, avidity, more readily, and/or with greater duration than its bindings to other targets.
  • an antibody that specifically recognizes an antigen reacts with one or more antigenic determinants of the antigen with a binding affinity that is at least about 10 times its binding affinity for other targets.
  • catabody refers to an antibody with catalytic activity.
  • a catabody can catalyze hydrolysis of the target antigen that it specifically recognizes.
  • exemplary catabodies include, but are not limited to, proteolytic antibodies.
  • Catabodies are also known as catalytic antibodies, Abzymes and Catmabs.
  • the term“SHD catabody” as used herein refers to a proteolytic catabody having an SHD motif in the light chain variable region (VL).
  • The“SHD motif’ refers to serine, histidine, and aspartate residues that together serve as the catalytic triad for peptide bond cleavage by the catabody.
  • the SHD motif is a non-linear motif, in which the serine, histidine and aspartate residues of the catalytic triad are not next to each other in the amino acid sequence.
  • An“isolated” antibody as used herein refers to an antibody that (1) is not associated with proteins found in nature, (2) is free of other proteins from the same source, (3) is expressed by a cell from a different species, or, (4) does not occur in nature.
  • isolated nucleic acid as used herein is intended to mean a nucleic acid of genomic, cDNA, or synthetic origin or some combination thereof, which by virtue of its origin the“isolated nucleic acid” (1) is not associated with all or a portion of a polynucleotide in which the“isolated nucleic acid” is found in nature, (2) is operably linked to a polynucleotide which it is not linked to in nature, or (3) does not occur in nature as part of a larger sequence.
  • CDR or“complementarity determining region” is intended to mean the non-contiguous antigen combining sites found within the variable region of both heavy and light chain polypeptides. These particular regions have been described by Kabat et al, J. Biol. Chem. 252:6609-6616 (1977); Kabat et al. , U.S. Dept of Health and Human Services,“Sequences of proteins of immunological interest” (1991); Chothia et al, J. Mol. Biol. 196:901-917 (1987); Al-Lazikani B. et al., J. Mol. Biol., 273: 927-948 (1997);
  • chimeric antibodies refer to antibodies in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit a biological activity of this invention (see U.S. Patent No. 4,816,567; and Morrison et al. , Proc. Natl. Acad. Sci. USA,
  • “Fv” is the minimum antibody fragment which contains a complete antigen- recognition and -binding site. This fragment consists of a dimer of one heavy- and one light- chain variable region domain in tight, non-covalent association. From the folding of these two domains emanate six hypervariable loops (3 loops each from the heavy and light chain) that contribute the amino acid residues for antigen binding and confer antigen binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three CDRs specific for an antigen) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site.
  • “Single-chain Fv,” also abbreviated as“sFv” or“scFv,” are antibody fragments that comprise the VH and VL antibody domains connected into a single polypeptide chain.
  • the scFv polypeptide further comprises a polypeptide linker between the VH and VL domains which enables the scFv to form the desired structure for antigen binding.
  • diabodies refers to small antibody fragments prepared by constructing scFv fragments (see preceding paragraph) typically with short linkers (such as about 5 to about 10 residues) between the VH and VL domains such that inter-chain but not intra-chain pairing of the V domains is achieved, resulting in a bivalent fragment, i.e., fragment having two antigen-binding sites.
  • Bispecific diabodies are heterodimers of two“crossover” scFv fragments in which the VH and VL domains of the two antibodies are present on different polypeptide chains.
  • Diabodies are described more fully in, for example, EP 404,097; WO 93/11161; and Hollinger et al, Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993).
  • humanized forms of non-human (e.g, rodent) antibodies are chimeric antibodies that contain minimal sequence derived from the non-human antibody.
  • humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervariable region (HVR) of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit or non-human primate having the desired antibody specificity, affinity, and capability.
  • donor antibody such as mouse, rat, rabbit or non-human primate having the desired antibody specificity, affinity, and capability.
  • framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues.
  • humanized antibodies can comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance.
  • the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non- human immunoglobulin and all or substantially all of the FRs are those of a human immunoglobulin sequence.
  • the humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human
  • Percent (%) amino acid sequence identity or“homology” with respect to the polypeptide and antibody (e.g., catabody) sequences identified herein is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the polypeptide being compared, after aligning the sequences considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN, Megalign (DNASTAR), or MUSCLE software.
  • % amino acid sequence identity values are generated using the sequence comparison computer program MUSCLE (Edgar, R.C., Nucleic Acids Research 32(5): 1792-1797, 2004; Edgar, R.C., BMC Bioinformatics 5(1):113, 2004).
  • an FcR of this invention is one that binds an IgG antibody (a g receptor) and includes receptors of the FcyRI, FcyRII, and FcyRIII subclasses, including allelic variants and alternatively spliced farms of these receptors.
  • FcyRII receptors include FcyRIIA (an“activating receptor”) and FcyRIIB (an“inhibiting receptor”), which have similar amino acid sequences that differ primarily in the cytoplasmic domains thereof.
  • Activating receptor FcyRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain.
  • Inhibiting receptor FcyRIIB contains an immunoreceptor tyrosine-based inhibition motif (GPM) in its cytoplasmic domain (see review M. in Daeron, Amu. Rev. Immunol. 15:203-234 (1997)).
  • ITAM immunoreceptor tyrosine-based activation motif
  • GPM immunoreceptor tyrosine-based inhibition motif
  • FcyRIIIA allotypes FcyRIIIA-Phel58, FcyRIIIA-Vall58, FcyRIIA-R131 and/or FcyRHA-H131.
  • FcRs are reviewed in Ravetch and Kinet, Amu. Rev. Immunol 9:457-92 (1991); Capel etal, Immunomethods 4:25-34 (1994); and de Haas etal, J. Lab. Clin. Med. 126:330-41 (1995).
  • Other FcRs including those to be identified in the future, are encompassed by the term“FcR” herein.
  • the term also includes the neonatal receptor, FcRn, which is responsible for the transfer of maternal IgGs to the fetus (Guyer et al, J Immunol. 117:587 (1976) and Kim et al, J Immunol. 24:249 (1994)).
  • FcRn neonatal receptor
  • FcRn refers to the neonatal Fc receptor (FcRn).
  • FcRn is structurally similar to major histocompatibility complex (MHC) and consists of an a-chain noncovalently bound to p2-microglobulin.
  • MHC major histocompatibility complex
  • FcRn plays a role in the passive delivery of immunoglobulin IgGs from mother to young and the regulation of serum IgG levels.
  • FcRn can act as a salvage receptor, binding and transporting pinocytosed IgGs in intact form both within and across cells, and rescuing them from a default degradative pathway.
  • The“CHI domain” of a human IgG Fc region (also referred to as“Cl” of“HI” domain) usually extends from about amino acid 118 to about amino acid 215 (EU numbering system).
  • Hinge region is generally defined as stretching from Glu216 to Pro230 of human IgGl (Burton, Molec. Immunol.22 ⁇ .161-206 (1985)). Hinge regions of other IgG isotypes may be aligned with the IgGl sequence by placing the first and last cysteine residues forming inter-heavy chain S-S bonds in the same positions.
  • The“CH2 domain” of a human IgG Fc region usually extends from about amino acid 231 to about amino acid 340.
  • the CH2 domain is unique in that it is not closely paired with another domain. Rather, two N-linked branched carbohydrate chains are interposed between the two CH2 domains of an intact native IgG molecule. It has been speculated that the carbohydrate may provide a substitute for the domain-domain pairing and help stabilize the CH2 domain.
  • The“CH3 domain” (also referred to as“C2” or‘ ⁇ 3” domain) comprises the stretch of residues C-terminal to a CH2 domain in an Fc region (i.e. from about amino acid residue 341 to the C-terminal end of an antibody sequence, typically at amino acid residue 446 or 447 of an IgG).
  • A“functional Fc fragment” possesses an“effector function” of a native sequence Fc region.
  • exemplary“effector functions” include Clq binding; complement dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down regulation of cell surface receptors (e.g. B cell receptor; BCR), etc.
  • Such effector functions generally require the Fc region to be combined with a binding domain (e.g . an antibody variable domain) and can be assessed using various assays known in the art.
  • An antibody with a variant IgG Fc with“altered” FcR binding affinity or ADCC activity is one which has either enhanced or diminished FcR binding activity (e.g., FcyR or FcRn) and/or ADCC activity compared to a parent polypeptide or to a polypeptide comprising a native sequence Fc region.
  • the variant Fc which“exhibits increased binding” to an FcR binds at least one FcR with higher affinity (e.g., lower apparent Kd or ICso value) than the parent polypeptide or a native sequence IgG Fc.
  • the improvement in binding compared to a parent polypeptide is about 3 fold, such as about any of 5, 10, 25, 50, 60, 100, 150, 200, or up to 500 fold, or about 25% to 1000% improvement in binding.
  • the polypeptide variant which“exhibits decreased binding” to an FcR binds at least one FcR with lower affinity (e.g. , higher apparent K d or higher ICso value) than a parent polypeptide.
  • the decrease in binding compared to a parent polypeptide may be about 40% or more decrease in binding.
  • “Antibody-dependent cell-mediated cytotoxicity” or“ADCC” refers to a form of cytotoxicity in which secreted Ig bound to Fc receptors (FcRs) present on certain cytotoxic cells (e.g, Natural Killer (NK) cells, neutrophils, and macrophages) enable these cytotoxic effector cells to bind specifically to an antigen-bearing target cell and subsequently kill the target cell with cytotoxins.
  • FcRs Fc receptors
  • cytotoxic cells e.g, Natural Killer (NK) cells, neutrophils, and macrophages
  • the primary cells for mediating ADCC NK cells, express FcyRHI only, w'hercas monocytes express FcyRI, FcyRII and FcyRIII.
  • ADCC activity of a molecule of interest is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991).
  • an in vitro ADCC assay such as that described in US Patent No. 5,500,362 or 5,821,337 may be performed.
  • Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and Natural Killer (NK) cells.
  • PBMC peripheral blood mononuclear cells
  • NK Natural Killer
  • ADCC activity of the molecule of interest may be assessed in vivo, e.g, in an animal model such as that disclosed in Clynes et al. PNAS (USA) 95:652-656 (1998).
  • the polypeptide comprising a variant Fc region which“exhibits increased ADCC” or mediates ADCC in the presence of human effector cells more effectively than a polypeptide having wild type IgG Fc or a parent polypeptide is one which in vitro or in vivo is substantially more effective at mediating ADCC, when the amounts of polypeptide wife variant Fc region and the polypeptide with wild type Fc region (or the parent polypeptide) in the assay are essentially the same.
  • such variants will be identified using any in vitro ADCC assay known in the art, such as assays or methods for determining ADCC activity, e.g., in an animal model etc.
  • the variant is from about 5 fold to about 100 fold, e.g. from about 25 to about 50 fold, more effective at mediating ADCC than the wild type Fc (or parent polypeptide) .
  • “Complement dependent cytotoxicity” or“CDC” refers to the lysis of a target cell in the presence of complement. Activation of the classical complement pathway is initiated by the binding of the first component of the complement system (Clq) to antibodies (of the appropriate subclass) which are bound to their cognate antigen.
  • a CDC assay e.g. as described in Gazzano-Santoro etai, J. Immunol. Methods 202: 163 (1996), may be performed.
  • Polypeptide variants with altered Fc region amino acid sequences and increased or decreased Clq binding capability are described in US patent No. 6,194,551B1 and W099/51642. The contents of those patent publications are specifically incorporated herein by reference. See also, Idusogie etal. J. Immunol. 164: 4178-4184
  • nucleotide sequence encoding an amino acid sequence includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence.
  • the phrase nucleotide sequence that encodes a protein or an RNA may also include introns to the extent that the nucleotide sequence encoding the protein may in some version contain an intron(s).
  • operably linked refers to functional linkage between a regulatory sequence and a heterologous nucleic acid sequence resulting in expression of the latter.
  • a first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence.
  • a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence.
  • operably linked DNA sequences are contiguous and, where necessary to join two protein coding regions, in the same reading frame.
  • “Homologous” refers to the sequence similarity' or sequence identity between two polypeptides or between two nucleic acid molecules. When a position in both of the two compared sequences is occupied by the same base or amino acid monomer subunit, e.g., if a position in each of two DNA molecules is occupied by adenine, then the molecules are homologous at that position.
  • the percent of homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions compared times 100. For example, if 6 of 10 of the positions in two sequences are matched or homologous then the two sequences are 60% homologous.
  • the DNA sequences ATTGCC and TATGGC share 50% homology. Generally, a comparison is made when two sequences are aligned to give maximum homology.
  • An“effective amount” of an antibody or composition as disclosed herein is an amount sufficient to carry out a specifically stated purpose.
  • An“effective amount” can be determined empirically and by known methods relating to the stated purpose.
  • pharmaceutically acceptable or“pharmacologically compatible” is meant a material that is not biologically or otherwise undesirable, e.g., the material may be incorporated into a pharmaceutical composition administered to a patient without causing any significant undesirable biological effects or interacting in a deleterious manner with any of the other components of the composition in which it is contained.
  • Pharmaceutically acceptable carriers or excipients have preferably met the required standards of toxicological and manufacturing testing and/or are included on the Inactive Ingredient Guide prepared by the U.S. Food and Drug administration.
  • Reference to“about” a value or parameter herein includes (and describes) variations that are directed to that value or parameter per se. For example, description referring to “about X” includes description of“X”.
  • reference to“not” a value or parameter generally means and describes “other than” a value or parameter.
  • the present application provides methods for determining levels of one or more catabodies (e g., SHD catabodies) in a sample, and methods for diagnosing (including determining a risk), treating or preventing a protein aggregation disease (PAD) in an individual.
  • the level of one or more catabodies e.g., SHD catabodies
  • the level of one or more catabodies is the protein level of the one or more catabodies.
  • the level of one or more catabodies is the mKNA level of the one or more catabodies.
  • the methods described herein use a substrate peptide in an immunoassay to detect binding of the substrate peptide with one or more catabodies (e.g, SHD catabodies) in a sample, thereby providing the level of one or more catabodies in the sample.
  • the level of one or more catabodies is the level of total catabodies, such as total SHD catabodies.
  • the level of one or more catabodies is the level of one or more (such as 1, 2, 3, 4, or more) catabodies (e.g., total SHD catabody levels) that specifically bind and cleave a target protein (e.g. , Ab) that is associated with a PAD.
  • the level of total SHD catabodies is determined based on binding of the catabodies to a substrate peptide comprising the amino acid sequence (EAR)n (SEQ ID NO: 2), wherein n is an integer between 1 and 30.
  • the substrate peptide may comprise any suitable number of EAR repeats, including, for example, about any one of, 1- 10, 10-20, 20-30, 1-30, 1-5, 5-10, 5-15, or 15-30.
  • the substrate peptide comprises the amino acid sequence SEQ ID NO: 2, wherein n is about any one of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.
  • the amino acid sequence of SEQ ID NO: 2 is at the N-terminus, C-terminus, or an internal position in the substrate peptide.
  • the substrate peptide comprises amino acid residues in addition to the amino acid sequence of SEQ ID NO: 2, e.g, at the N-terminus and/or C-terminus of the amino acid sequence of SEQ ID NO:2. In some embodiments, the substrate peptide comprises at least about any one of 1 , 2, 3, 5, 10, 15, 20, 25, or 30 amino acids in addition to the amino acid sequence of SEQ ID NO: 2.
  • the substrate peptide has a total length of about 3-100 amino acids, such as about any one of 3-10, 10-20, 20-30, 30-40, 40-50, 50-60, 60-70, 70-80, 80-90, 90-100, 3-50, 50-100, 10-30, 30-60, 60-90, 20-40, 40-60, 60-80 or 80- 100 amino acids.
  • the substrate peptide comprises a label, e.g, a fluorescent label, a peptide tag, or a biotin label.
  • the substrate peptide comprises the formula EAR-AMC (SEQ ID NO: 1).
  • EAR-AMC i.e., a peptide having the amino acid sequence EAR conjugated to 7-amino-4-methylcoumarin (AMC)
  • AMC 7-amino-4-methylcoumarin
  • a substrate peptide comprising the amino acid sequence of SEQ ID NO: 2 has not been used to measure the binding activity and levels of catabodies in a biological sample. It is also unpredictable whether the binding between the EAR peptides and the SHD catabodies is strong and long-lasting enough to allow accurate determination of the level of SHD catabodies via a binding assay.
  • the present application provides an immunoassay using a substrate peptide comprising the amino acid sequence of SEQ ID NO: 2, which provides accurate readout for the level of total SHD catabodies in a biological sample (e.g ., serum sample).
  • a method for determining the level of one or more SHD catabodies in a biological sample comprising: a) contacting the biological sample with a substrate peptide immobilized on a solid support under conditions that allow formation of a catabody-substrate peptide complex, and b) determining the amount of the catabody-substrate peptide complex, thereby providing the level of one or more SHD catabodies in the biological sample, wherein the substrate peptide comprises the amino acid sequence (EAR)n (SEQ ID NO: 2), and wherein n is an integer between 1 and 30. In some embodiments, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.
  • the substrate peptide has the formula EAR-AMC (SEQ ID NO: 1). In some embodiments, the substrate peptide comprises the amino acid sequence EAREAREAR (SEQ ID NO: 3). In some embodiments, the solid support is an ELISA plate.
  • a method for determining the level of one or more SHD catabodies in a biological sample comprising: a) contacting the biological sample with a substrate peptide immobilized on a solid support under conditions that allow formation of a catabody-substrate peptide complex, and b) determining the amount of the catabody- substrate peptide complex using an antibody that specifically binds to total Ig (e.g., total human Ig, such as total IgM, total IgG, total IgA, and/or total IgE), thereby providing the level of one or more SHD catabodies in the biological sample, wherein the substrate peptide comprises the amino acid sequence (EAR)n (SEQ ID NO: 2), and wherein n is an integer between 1 and 30 (e.g., n is 3).
  • the antibody is labeled with an enzyme (e.g, HRP) or a fluorescent label (e.g, FITC).
  • an enzyme e.g, HRP
  • FITC fluorescent label
  • a method for determining the level of one or more SHD catabodies in a biological sample comprising: a) contacting the biological sample with a substrate peptide immobilized on a solid support under conditions that allow formation of a catabody-substrate peptide complex, and b) determining the amount of the catabody- substrate peptide complex using an antibody that specifically binds to total Ig (e.g., total human Ig, such as total IgM, total IgG, total IgA, and/or total IgE), thereby providing the level of one or more SHD catabodies in the biological sample, wherein the substrate peptide comprises the amino acid sequence of SEQ ID NO: 3.
  • the antibody is labeled with an enzyme (e.g., HRP) or a fluorescent label (e.g, FITC).
  • the solid support is an ELISA plate.
  • a method for determining the level of one or more SHD catabodies in a biological sample comprising: a) contacting a substrate peptide with an ELISA plate to coat the wells of the ELISA plate with the substrate peptide; b) contacting the biological sample with a coated well of the ELISA plate under conditions that allow formation of a catabody-substrate peptide complex, and b) determining the amount of the catabody-substrate peptide complex using an antibody that specifically binds to total Ig (e.g., total human Ig, such as total IgM, total IgG, total IgA, and/or total IgE) labeled with an enzyme (e.g, HRP) or a fluorescent label (e.g, FITC), thereby providing the level of one or more SHD catabodies in the biological sample, wherein the substrate peptide comprises the amino acid sequence of SEQ ID NO: 3.
  • a method for determining a risk for a protein aggregation disease (PAD) in an individual, wherein the PAD is associated with aggregation of a target protein comprising determining the level of one or more SHD catabodies in a biological sample (e.g., serum sample) of the individual, wherein the individual is determined as having a risk for the PAD if the level of the one or more SHD catabodies is lower than a control SHD catabody level.
  • the level of one or more SHD catabodies is the level of one or more SHD catabodies that specifically bind to the target protein.
  • the level of one or more SHD catabodies is the level of total SHD catabodies. In some embodiments, the level of total SHD catabodies is determined by contacting a semm sample of tire individual with a substrate peptide under conditions that allow formation of a catabody-substrate peptide complex, and determining the amount of the catabody-substrate peptide complex. In some embodiments, the substrate peptide is immobilized on a solid support. In some embodiments, the level of the one or more SHD catabodies is determined using any one of the methods of determining SHD catabody levels described herein.
  • the PAD is Alzheimer’s disease, and the target protein is Ab;
  • the PAD is Parkinson’s disease, and the target protein is a-synuclein;
  • the PAD is Alzheimer’s disease or dementia, and the target protein is Tau;
  • the PAD is ATTR amyloidosis, and the target protein is transthyretin;
  • the PAD is AL amyloidosis, and the target protein is immunoglobulin light chain;
  • the PAD is FTLD or ALS, and the target protein is TDP43;
  • the PAD is Huntington’s disease, and the target protein is Huntingtin;
  • the PAD is Type P diabetes, and the target protein is LAPP; and
  • the PAD is ALS, and the target protein is SOD1.
  • AD Alzheimer’s disease in an individual, comprising: a) contacting a semm sample from the individual with a substrate peptide immobilized on a solid support under conditions that allow formation of a catabody-substrate peptide complex, and b) determining the amount of one or more SHD catabodies that specifically bind to Ab, and wherein the individual is determined as having a risk for the AD if the level of one or more SHD catabodies is lower than a control SHD catabody level.
  • a method for determining a risk for a PAD in an individual comprising a) determining the level of one or more SHD catabodies in a biological sample (e.g., serum sample) of the individual, and b) determining the level of an auto-antibody against the target protein in a biological sample (e.g., serum sample) of the individual, and wherein the individual is determined as having a risk for the PAD if: (i) the level of the one or more SHD catabodies is lower than a control SHD catabody level; and (ii) the level of the auto-antibody against the target protein is lower than a control auto-antibody level.
  • the level of one or more SHD catabodies is the level of one or more SHD catabodies that specifically bind to the target protein. In some embodiments, the level of one or mote SHD catabodies is the level of total SHD catabodies. In some embodiments, the level of total SHD catabodies is determined by contacting a serum sample of the individual with a substrate peptide under conditions that allow formation of a catabody-substrate peptide complex, and determining the amount of the catabody-substrate peptide complex. In some embodiments, the substrate peptide is immobilized on a solid support.
  • the level of the one or more SHD catabodies is determined using any one of the methods of determining SHD catabody levels described herein.
  • the level of the auto-antibody is determined by contacting a serum sample of the individual with the target protein (or a fragment thereof) under conditions that allow formation of an auto-antibody-target protein complex, and determining the amount of the auto-antibody-target protein complex.
  • the level of the auto-antibody is determined using an ELISA assay.
  • the PAD is Alzheimer’s disease, and the target protein is Ab;
  • the PAD is Parkinson’s disease, and the target protein is a-synuclein;
  • the PAD is Alzheimer’s disease or dementia, and the target protein is Tau;
  • the PAD is ATTR amyloidosis, and the target protein is transthyretin;
  • the PAD is AL amyloidosis, and the target protein is immunoglobulin light chain;
  • the PAD is FTLD or ALS, and the target protein is TDP43;
  • the PAD is Huntington’s disease, and the target protein is Huntingtin;
  • the PAD is Type P diabetes, and the target protein is IAPP; and
  • the PAD is ALS, and the target protein is SOD1.
  • a method for determining a risk for a PAD in an individual comprising a) determining the level of one or more SHD catabodies in a biological sample (e.g., serum sample) of the individual, and b) determining the level of the target protein in a biological sample (e.g., semm sample or cerebrospinal fluid sample) of the individual, and wherein the individual is determined as having a risk for the PAD if: (i) tire level of the one or more SHD catabodies is lower than a control SHD catabody level; and (ii) the level of the target protein is higher than a control target protein level.
  • a biological sample e.g., serum sample
  • a biological sample e.g., semm sample or cerebrospinal fluid sample
  • the level of one or more SHD catabodies is the level of one or more SHD catabodies that specifically bind to the target protein. In some embodiments, the level of one or mote SHD catabodies is the level of total SHD catabodies. In some embodiments, the level of total SHD catabodies is determined by contacting a serum sample of the individual with a substrate peptide under conditions that allow formation of a catabody-substrate peptide complex, and determining the amount of the catabody-substrate peptide complex. In some embodiments, the substrate peptide is immobilized on a solid support.
  • the level of the one or more SHD catabodies is determined using any one of the methods of determining SHD catabody levels described herein.
  • the level of the target protein is determined by contacting the biological sample of the individual with an antibody against the target protein under conditions that allow formation of an antibody-target protein complex, and determining the amount of the antibody-target protein complex.
  • the level of the target protein is determined using an ELISA assay.
  • the PAD is Alzheimer’s disease, and the target protein is Ab;
  • the PAD is Parkinson’s disease, and the target protein is a-synuclein;
  • the PAD is Alzheimer’s disease or dementia, and the target protein is Tau;
  • the PAD is ATTR amyloidosis, and the target protein is transthyretin;
  • the PAD is AL amyloidosis, and the target protein is immunoglobulin light chain;
  • the PAD is FTLD or ALS, and the target protein is TDP43;
  • the PAD is Huntington’s disease, and the target protein is Huntingtin;
  • the PAD is Type II diabetes, and the target protein is IAPP; and
  • the PAD is ALS, and the target protein is SOD1.
  • a method for determining a risk for a PAD in an individual comprising: a) contacting a serum sample of the individual with a substrate peptide immobilized on a solid support under conditions that allow formation of a catabody-substrate peptide complex, wherein the substrate peptide comprises the amino acid sequence (EAR) n (SEQ ID NO: 2), wherein n is an integer between 1 and 30 (e.g., n is 3); b) determining the amount of the catabody-substrate peptide complex, thereby providing the level of one or more SHD catabodies in the serum of the individual; and c) determining the level of an autoantibody against the target protein in a biological sample (e.g., serum sample) of the individual, and wherein the individual is determined as having a risk for the PAD if: (i) the level of the one or more SHD
  • the solid support is an ELISA plate.
  • the level of the auto-antibody is determined by contacting a serum sample of the individual with the target protein (or a fragment thereof) under conditions that allow formation of an auto- antibody-target protein complex, and determining the amount of the auto-antibody-target protein complex. In some embodiments, the level of the auto-antibody is determined using an ELISA assay.
  • the PAD is Alzheimer’s disease, and the target protein is Ap;
  • the PAD is Parkinson’s disease, and the target protein is a-synuclein;
  • the PAD is Alzheimer’s disease or dementia, and the target protein is Tau;
  • the PAD is ATTR amyloidosis, and the target protein is transthyretin;
  • the PAD is AL amyloidosis, and the target protein is immunoglobulin light chain;
  • the PAD is FTLD or ALS, and the target protein is TDP43;
  • the PAD is Huntington’s disease, and the target protein is Huntingtin;
  • the PAD is Type II diabetes, and the target protein is IAPP; and
  • the PAD is ALS, and the target protein is SOD1.
  • a method for determining a risk for a PAD in an individual comprising: a) contacting a serum sample of the individual with a substrate peptide immobilized on a solid support under conditions that allow formation of a catabody-substrate peptide complex, wherein the substrate peptide comprises the amino acid sequence (EAR)n (SEQ ID NO: 2), wherein n is an integer between 1 and 30 (e.g., n is 3); b) determining the amount of the catabody-substrate peptide complex, thereby providing the level of one or more SHD catabodies in the serum of the individual; and c) determining the level of the target protein in a biological sample (e.g, serum sample or cerebrospinal fluid sample) of the individual, and wherein the individual is determined as having a risk for the PAD if: (i) the level of the one or more S
  • the solid support is an ELISA plate.
  • the level of the target protein is determined by contacting the biological sample of the individual with an antibody against the target protein under conditions that allow formation of an antibody- target protein complex, and determining tire amount of the antibody-target protein complex.
  • the level of the target protein is determined using an ELISA assay.
  • the PAD is Alzheimer’s disease, and the target protein is Ab;
  • the PAD is Parkinson’s disease, and the target protein is a-synuclein;
  • the PAD is
  • the target protein is Tau
  • the PAD is ATTR amyloidosis, and the target protein is transthyretin
  • the PAD is AL amyloidosis, and the target protein is immunoglobulin light chain
  • the PAD is FTLD or ALS, and the target protein is ⁇ R43
  • the PAD is Huntington’s disease, and the target protein is Huntingtin
  • the PAD is Type II diabetes, and the target protein is LAPP
  • the PAD is ALS, and the target protein is SOD1.
  • a method for determining a risk for a PAD in an individual comprising: a) contacting a serum sample from the individual with a substrate peptide immobilized on a solid support under conditions that allow formation of a catabody-substrate peptide complex, wherein the substrate peptide comprises the amino acid sequence (EAR) n (SEQ ID NO: 2), wherein n is an integer between 1 and 30 (e.g., n is 3); b) determining the amount of the catabody-substrate peptide complex using an antibody that specifically binds to total Ig (e.g, total human Ig, such as total IgM, total IgG, total IgA, and/or total IgE), thereby providing the level of one or more SHD catabodies in the serum of the individual; and c) determining the level of an auto-antibody against the target protein in
  • tire antibody specifically binding to Ig is labeled with an enzyme (e.g, HRP) or a fluorescent label (e.g, FITC).
  • the solid support is an ELISA plate.
  • the level of the auto-antibody is determined by contacting a serum sample of the individual with the target protein (or a fragment thereof) under conditions that allow formation of an auto-antibody- target protein complex, and determining the amount of the auto-antibody-target protein complex. In some embodiments, the level of the auto-antibody is determined using an ELISA assay.
  • the PAD is Alzheimer’s disease, and the target protein is Ab;
  • the PAD is Parkinson’s disease, and the target protein is a-synuclein;
  • the PAD is Alzheimer’s disease or dementia, and the target protein is Tau;
  • the PAD is ATTR amyloidosis, and the target protein is transthyretin;
  • the PAD is AL amyloidosis, and the target protein is immunoglobulin light chain;
  • the PAD is FTLD or ALS, and the target protein is TDP43;
  • the PAD is Huntington’s disease, and the target protein is Huntingtin;
  • the PAD is Type II diabetes, and the target protein is LAPP; and
  • the PAD is ALS, and the target protein is SOD1.
  • a method for determining a risk for a PAD in an individual comprising: a) contacting a serum sample from the individual with a substrate peptide immobilized on a solid support under conditions that allow formation of a catabody-substrate peptide complex, wherein the substrate peptide comprises the amino acid sequence (EAR) n (SEQ ID NO: 2), wherein n is an integer between 1 and 30 (e.g, n is 3); b) determining the amount of the catabody-substrate peptide complex using an antibody that specifically binds to total Ig (e.g., total human Ig, such as total IgM, total IgG, total IgA, and/or total IgE), thereby providing the level of one or more SHD catabodies in the serum of the individual; and c) determining the level of the target protein in a biological sample (e.
  • the solid support is an ELISA plate.
  • the level of the target protein is determined by contacting the biological sample of the individual with an antibody against the target protein under conditions that allow formation of an antibody-target protein complex, and determining the amount of the antibody-target protein complex. In some embodiments, the level of the target protein is determined using an ELISA assay.
  • the PAD is Alzheimer’s disease, and the target protein is Ab;
  • the PAD is Parkinson’s disease, and the target protein is a- synuclein;
  • the PAD is Alzheimer’s disease or dementia, and the target protein is Tau;
  • the PAD is ATTR amyloidosis, and the target protein is transthyretin;
  • the PAD is AL amyloidosis, and the target protein is immunoglobulin light chain;
  • the PAD is FTLD or ALS, and the target protein is TDP43;
  • the PAD is Huntington’s disease, and the target protein is Huntingtin;
  • the PAD is Type II diabetes, and the target protein is IAPP; and
  • the PAD is ALS, and the target protein is SOD1.
  • AD Alzheimer’s disease in an individual, comprising: a) contacting a serum sample from the individual with a substrate peptide immobilized on a solid support under conditions that allow formation of a catabody-substrate peptide complex, wherein the substrate peptide comprises the amino acid sequence of SEQ ID NO: 3; b) determining the amount of the catabody-substrate peptide complex using an antibody that specifically binds to total Ig (e.g., total human Ig, such as total IgM, total IgG, total IgA, and/or total IgE), thereby providing the level of one or more SHD catabodies in the serum of the individual,; c) contacting a semm sample of the individual with Ab (e.g., Ab(1-42)) under conditions that allow formation of an auto-antibody-Ab complex, and d) determining the amount of the auto- antibody-Ab complex, thereby providing the level of the auto-antibody against Ab; and wherein the individual is determined as having a
  • the antibody specifically binding to Ig is labeled with an enzyme (e.g., HRP) or a fluorescent label (e.g., FITC).
  • the solid support is an ELISA plate.
  • the level of the auto-antibody is determined using an ELISA assay.
  • AD Alzheimer’s disease in an individual, comprising: a) contacting a serum sample from the individual with a substrate peptide immobilized on a solid support under conditions that allow formation of a catabody-substrate peptide complex, wherein the substrate peptide comprises the amino acid sequence of SEQ ID NO: 3; b) determining the amount of the catabody-substrate peptide complex using an antibody that specifically binds to total Ig (e.g, total human Ig, such as total IgM, total IgG, total IgA, and/or total IgE), thereby providing the level of one or mote SHD catabodies in the serum of the individual,; c) contacting a cerebrospinal fluid sample of the individual with an anti-Ab antibody under conditions that allow formation of an antibody -Ab complex, and d) determining the amount of the antibody- Ab complex, thereby providing the level of Ab; and wherein the individual is determined as having a risk for the AD if: (i) the level of
  • the antibody specifically binding to Ig is labeled with an enzyme (e.g, HRP) or a fluorescent label (e.g., FITC).
  • the solid support is an ELISA plate.
  • the level of Ab is determined using an ELISA assay.
  • diagnosis methods described herein may be used to inform treatment of the PAD using any known therapeutic agents for treating the PAD in the art, or any catabodies (such as anti-Ab catabodies) described herein.
  • the diagnosis methods described herein allow early detection of a risk of PAD in an individual, thereby allowing early intervention and prophylactic treatment of the PAD.
  • a method of treating or preventing a PAD in an individual, wherein the PAD is associated with aggregation of a target protein comprising: a) determining the individual as having a risk for the PAD according to any one of the methods of determining a risk as described herein; and b) administering to the individual an effective amount of a therapeutic agent that treats the PAD.
  • a method of treating or preventing a PAD in an individual, wherein the PAD is associated with aggregation of a target protein comprising: a) determining the individual as having a risk for the PAD according to any one of the methods of determining a risk as described herein; and b) administering to the individual an effective amount of a therapeutic catabody that specifically binds to the target protein.
  • the method is repeated at a frequency of no more than about every three months, e.g, about every three months, about every six months, or about every year. In some embodiments, the method is carried out only once.
  • a method of treating or preventing AD in an individual comprising: a) determining the individual as having a risk for AD according to any one of the methods of determining a risk as described herein; and b) administering to the individual an effective amount of an anti-Ab catabody, such as any one of the anti-Ab catabodies described in Section IP.
  • the method is repeated at a frequency of no more than about every three months, e.g, about every three months, about every six months, or about every year.
  • a method of treating or preventing a PAD in an individual, wherein the PAD is associated with aggregation of a target protein comprising: a) determining the level of one or more SHD catabodies in a biological sample (e.g, serum sample) of the individual, wherein the individual is determined as having a risk for the PAD if the level of the one or more SHD catabodies is lower than a control SHD catabody level; and b) administering to the individual an effective amount of a therapeutic catabody that specifically binds to the taiget protein.
  • a biological sample e.g, serum sample
  • the level of one or more SHD catabodies is the level of one or more SHD catabodies that specifically bind to the target protein. In some embodiments, the level of one or more SHD catabodies is the level of total SHD catabodies. In some embodiments, the level of total SHD catabodies is determined by contacting a serum sample of the individual with a substrate peptide under conditions that allow formation of a catabody-substrate peptide complex, and determining the amount of the catabody-substrate peptide complex. In some embodiments, the substrate peptide is immobilized on a solid support.
  • the PAD is Alzheimer’s disease, and the target protein is Ab;
  • the PAD is Parkinson’s disease, and the target protein is a-synuclein;
  • the PAD is Alzheimer’s disease or dementia, and the target protein is Tau;
  • the PAD is ATTR amyloidosis, and the target protein is transthyretin;
  • the PAD is AL amyloidosis, and the taiget protein is immunoglobulin light chain;
  • the PAD is FILD or ALS, and the target protein is TDP43;
  • the PAD is Huntington’s disease, and the target protein is Huntingtin;
  • the PAD is Type P diabetes, and the taiget protein is IAPP; and
  • the PAD is ALS, and the taiget protein is SOD1.
  • the method is repeated at a frequency of no more than about every three months, e.g.,
  • a method of treating or preventing AD in an individual comprising: a) contacting a serum sample from the individual with a substrate peptide immobilized on a solid support under conditions that allow formation of a catabody- substrate peptide complex, and b) determining the amount of one or more SHD catabodies that specifically bind to Ab, and c) administering to the individual an effective amount of an anti-Ab catabody, if the level of the one or more SHD catabodies is lower than a control SHD catabody level.
  • the method is repeated at a frequency of no more than about every three months, e.g, about every three months, about every six months, or about every year.
  • a method of treating or preventing a PAD in an individual comprising: a) determining the level of one or more SHD catabodies in a biological sample (e.g, serum sample) of the individual, b) determining the level of an auto-antibody against the target protein a biological sample (e.g., serum sample) of the individual, and c) administering to the individual an effective amount of a therapeutic catabody that specifically binds to the target protein if: (i) the level of the one or more SHD catabodies is lower than a control SHD catabody level; and (ii) the level of the auto-antibody against the target protein is lower than a control auto-antibody level.
  • the level of one or more SHD catabodies is the level of one or more SHD catabodies that specifically bind to the target protein. In some embodiments, the level of one or more SHD catabodies is the level of total SHD catabodies. In some embodiments, the level of total catabodies is determined by contacting a serum sample of the individual with a substrate peptide under conditions that allow formation of a catabody-substrate peptide complex, and determining the amount of the catabody-substrate peptide complex. In some embodiments, the substrate peptide is immobilized on a solid support.
  • the level of the auto-antibody is determined by contacting a serum sample of the individual with the target protein under conditions that allow formation of an auto-antibody-target protein complex, and determining the amount of the auto-antibody-target protein complex. In some embodiments, the level of the auto-antibody is determined using an ELISA assay.
  • the PAD is Alzheimer’s disease, and the target protein is Ab;
  • the PAD is Parkinson’s disease, and the target protein is a-synuclein;
  • the PAD is Alzheimer’s disease or dementia, and the target protein is Tau;
  • the PAD is ATTR amyloidosis, and the target protein is transthyretin;
  • the PAD is AL amyloidosis, and the target protein is immunoglobulin light chain;
  • the PAD is FTLD or ALS, and the target protein is TDP43;
  • the PAD is Huntington’s disease, and the target protein is Huntingtin;
  • the PAD is Type II diabetes, and the target protein is IAPP; and
  • the PAD is ALS, and the target protein is SOD1.
  • the method is repeated at a frequency of no more than about every three months, e.g., about every three months, about every six months, or about
  • a method of treating or preventing a PAD in an individual, wherein the PAD is associated with aggregation of a target protein comprising: a) contacting a serum sample of the individual with a substrate peptide immobilized on a solid support under conditions that allow formation of a catabody-substrate peptide complex, wherein the substrate peptide comprises the amino acid sequence (EAR) n (SEQ ID NO: 2), wherein n is an integer between 1 and 30 (e.g., n is 3); b) determining the amount of the catabody-substrate peptide complex, thereby providing the level of one or more SHD catabodies in the serum of the individual; c) determining the level of an auto- antibody against the target protein in a biological sample (e.g., serum sample) of the individual, and d) administering to the individual an effective amount of a therapeutic catabody that specifically binds to the taiget protein if
  • the amount of the catabody -substrate peptide complex is determined using an antibody that specifically binds to total Ig (e.g., total human Ig, such as total IgM, total IgG, total IgA, and/or total IgE).
  • the antibody specifically binding to Ig is labeled with an enzyme (e.g., HRP) or a fluorescent label (e.g., FITC).
  • the solid support is an ELISA plate.
  • the level of the auto-antibody is determined by contacting a serum sample of the individual with the taiget protein under conditions that allow formation of an auto-antibody-target protein complex, and determining the amount of the auto-antibody-target protein complex. In some embodiments, the level of the auto-antibody is determined using an ELISA assay.
  • the PAD is Alzheimer’s disease, and the taiget protein is Ab;
  • the PAD is Parkinson’s disease, and the target protein is a-synuclein;
  • the PAD is Alzheimer’s disease or dementia, and the target protein is Tau;
  • the PAD is AT I K amyloidosis, and the taiget protein is transthyretin;
  • the PAD is AL amyloidosis, and the taiget protein is immunoglobulin tight chain;
  • the PAD is FTLD or ALS, and the target protein is TDP43;
  • the PAD is Huntington’s disease, and the taiget protein is Huntingtin;
  • the PAD is Type II diabetes, and the taiget protein is IAPP; and
  • the PAD is ALS, and the taiget protein is SOD1.
  • the method is repeated at a frequency of no
  • AD Alzheimer’s disease in an individual, comprising: a) contacting a serum sample from the individual with a substrate peptide immobilized on a solid support under conditions that allow formation of a catabody-substrate peptide complex, wherein the substrate peptide comprises the amino acid sequence of SEQ ID NO: 3; b) determining the amount of the catabody-substrate peptide complex using an antibody that specifically binds to total lg (e.g, total human Ig, such as total IgM, total IgG, total IgA, and/or total IgE), thereby providing the level of one or more SHD catabodies in the serum of the individual; c) contacting a serum sample of the individual with Ab (e.g., Ab(1-42)) under conditions that allow formation of an auto-antibody-Ab complex, d) determining the amount of the auto-antibody -Ab complex using an antibody that specifically binds to total Ig (e.g., total human Ig, such as total I
  • the antibody specifically binding to Ig is labeled with an enzyme (e.g., HRP) or a fluorescent label (e.g, FITC).
  • the solid support is an ELISA plate.
  • the method is repeated at a frequency of no more than about every three months, e.g, about every three months, about every six months, or about every year.
  • AD Alzheimer’s disease in an individual, comprising: a) determining the level of one or more SHD catabodies in a biological sample (e.g, serum sample) of the individual, b) determining the level of an auto-antibody against Ab in a biological sample (e.g, serum sample) of the individual, and c) administering to the individual an effective amount of an anti-Ab catabody, if: (i) the level of the one or more SHD catabodies is lower than a control SHD catabody level; and (ii) the level of the auto-antibody against Ab is lower than a control auto-antibody level, wherein the anti-Ab catabody comprises: a light chain variable region (VL) comprising a light chain complementarity determining region (LC-CDR) 1 comprising the amino acid sequence of SEQ ID NO: 12, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 13, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 14, or a
  • the anti-Ab catabody comprises: a heavy chain variable region (VH) comprising a heavy chain complementarity determining region (HC-CDR) 1 comprising the amino acid sequence of SEQ ID NO: 9, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 10, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 11, or a variant thereof comprising up to about 5 amino acid substitutions in the HC-CDRs.
  • VH heavy chain variable region
  • HC-CDR heavy chain complementarity determining region
  • the amino acid residue at position 26 of the VL is S
  • the amino acid residue at position 27D of the VL is D, E or H
  • the amino acid residue at position 28 of the VL is D or N
  • the numbering is according to the EU index of Rabat.
  • the anti-Ab catabody comprises: a VH comprising an amino acid sequence having at least about 85% sequence identity to the amino acid sequence of SEQ ID NO: 4, 6, 19 or 20; and/or a VL comprising an amino acid sequence having at least about 85% sequence identity to the amino acid sequence of SEQ ID NO: 5, 7, 8, 21 or 22.
  • the anti-Ab catabody comprises: (i) a VH comprising the amino acid sequence of SEQ ID NO: 4, and a VL comprising the amino acid sequence of SEQ ID NO: 5; (ii) a VH comprising the amino acid sequence of SEQ ID NO: 6, and a VL comprising the amino acid sequence of SEQ ID NO: 7; (iii) a VH comprising the amino acid sequence of SEQ ID NO: 6, and a VL comprising the amino acid sequence of SEQ ID NO: 8; (iv) a VH comprising the amino acid sequence of SEQ ID NO: 19, and a VL comprising the amino acid sequence of SEQ ID NO: 21 ; (v) a VH comprising the amino acid sequence of SEQ ID NO: 20, and a VL comprising the amino acid sequence of SEQ ID NO: 21; (vi) a VH comprising the amino acid sequence of SEQ ID NO: 19, and a VL comprising the amino acid sequence of SEQ ID NO: 22; or
  • the anti-Ab catabody is a full-length antibody, such as an IgGl or lgG4 antibody.
  • the method is repeated at a frequency of no more than about every three months, e.g. , about every three months, about every six months, or about every year.
  • a method of treating or preventing AD in an individual comprising: a) contacting a serum sample from the individual with a substrate peptide immobilized on a solid support under conditions that allow formation of a catabody- substrate peptide complex, wherein the substrate peptide comprises the amino acid sequence of SEQ P) NO: 3; b) determining the amount of the catabody-substrate peptide complex using an antibody that specifically binds to total Ig (e.g., total human Ig, such as total IgM, total IgG, total IgA, and/or total IgE), thereby providing the level of one or more SHD catabodies in the serum of the individual; c) contacting a serum sample of the individual with Ab (e.g, Ab(1-42)) under conditions that allow formation of an auto-anti body-Ab complex, d) determining the amount of the auto-anti body-Ab complex using an antibody that specifically binds to total Ig (e.g.
  • the amino acid residue at position 26 of the VL is S
  • the amino acid residue at position 27D of the VL is D, E or H
  • the amino acid residue at position 28 of the VL is D or N
  • the numbering is according to the EU index of Rabat.
  • the anti-Ab catabody comprises: a VH comprising an amino acid sequence having at least about 85% sequence identity to the amino acid sequence of SEQ ID NO: 4, 6, 19 or 20; and/or a VL comprising an amino acid sequence having at least about 85% sequence identity to the amino acid sequence of SEQ ID NO: 5, 7, 8, 21 or 22.
  • the anti-Ab catabody comprises: (i) a VH comprising the amino acid sequence of SEQ ID NO: 4, and a VL comprising the amino acid sequence of SEQ ID NO: 5; (ii) a VH comprising the amino acid sequence of SEQ ID NO: 6, and a VL comprising the amino acid sequence of SEQ ID NO: 7; (iii) a VH comprising the amino acid sequence of SEQ ID NO: 6, and a VL comprising the amino acid sequence of SEQ ID NO: 8; (iv) a VH comprising the amino acid sequence of SEQ ID NO: 19, and a VL comprising the amino acid sequence of SEQ ID NO: 21; (v) a VH comprising the amino acid sequence of SEQ ID NO: 20, and a VL comprising the amino acid sequence of SEQ ID NO: 21; (vi) a VH comprising the amino acid sequence of SEQ ID NO: 19, and a VL comprising the amino acid sequence of SEQ ID NO: 22; or (vii) a V
  • the anti-Ab catabody is a full-length antibody, such as an IgGl or IgG4 antibody.
  • the antibody specifically binding to lg is labeled with an enzyme (e.g, HRP) or a fluorescent label (e.g, FITC).
  • the solid support is an ELISA plate.
  • the method is repeated at a frequency of no more than about every' three months, e.g. , about every three months, about every six months, or about every year.
  • the methods described herein detect the levels of one or more SHD catabodies, autoantibodies against target proteins, and target proteins, including, but not limited to, protein levels and mRNA levels. Protein levels may be detected using immunoassays, mass spectroscopy or other molecular biology techniques. Levels of mRNA may be detected using quantitative PCR or other molecular biology' techniques.
  • the method comprises detecting the level of one or more SHD catabodies using a substrate peptide comprising the amino acid sequence of SEQ ID NO: 2.
  • the level of one or more SHD catabodies is the level of total SHD catabodies. In some embodiments, the level of one or more SHD catabodies is the level of one or more SHD catabodies that specifically bind to the target protein. In some
  • the level of one or more SHD catabodies specifically binding to the target protein is determined by: a) extracting (e.g., immunologically pulling down) antibodies that specifically bind to the target protein from the biological sample, and b) contacting the extracted antibodies with the substrate peptide.
  • the level of one or more SHD catabodies that specifically bind to the target protein is determined by: a) contacting the biological sample with a target protein (e.g, Ab) immobilized on a solid support under conditions that allow formation of a catabody-target protein complex; b) contacting the catabody-target protein complex with a substrate peptide comprising a label (e.g.
  • AMC or biotin under conditions that allow formation of a catabody-target protein- substrate peptide complex; c) contacting the catabody-target protein-substrate peptide with an antibody against the label; and d) determining the amount of antibody against the label bound to the catabody-target protein-substrate peptide, thereby providing the level of one or more SHD catabodies that specifically bind to the target protein.
  • the substrate peptide or the target protein (or fragment thereof, e.g. , Ab) may be obtained by chemical synthesis.
  • the substrate peptide or the target protein (or fragment thereof, e.g, Ab) may be immobilized to a solid support via an immobilization moiety such as biotin, streptavidin, avidin, or a peptide tag.
  • the solid support is functionalized for conjugation with the substrate peptide.
  • the solid support is an ELISA plate.
  • the ELISA plate may be a flat-bottomed, multi-well (e.g, 96- well) plates, made from polystyrene or polyvinyl chloride.
  • the substrate peptide or the target protein (or fragment thereof, e.g, Ab) may be coated onto an ELISA plate via passive adsorption. Adsorption occurs passively as the result of hydrophobic interactions between the amino acids side chains on the substrate peptides or the target protein (or fragment thereof, e.g., Ab) and the plastic surface of the ELISA plate.
  • the substrate peptide is coated on the surface of an ELISA plate at the density of about 1-2 pg/well.
  • Exemplary' coating conditions on an ELISA plate involve adding 50-100 m ⁇ of coating buffer, containing the substrate peptide at a concentration of 1-10 pg/ml, and incubating overnight at 4 °C or for 1-3 hours at 37 °C.
  • Alternative temperatures, times, buffers, and coating agent concentrations can be used and should be tested by experimentation.
  • Exemplary coating buffers include bicarbonate buffer at pH 9.6 and phosphate buffer saline (PBS).
  • the solid support e.g., ELISA plate
  • awash buffer such as PBS or PBST (0.1% TWEEN-20 in PBS).
  • the solid support e.g, ELISA plate
  • a blocking buffer such as 10% fetal bovine serum (FBS) in PBS, or 1% BSA in PBS.
  • FBS fetal bovine serum
  • the solid support e.g, ELISA plate
  • a wash buffer such as PBS or PBST.
  • the levels of one or more catabodies, auto-antibodies against the target protein and the target protein are determined using a sample (e.g, a sample from an individual or a reference sample).
  • the sample is a biological sample.
  • the biological sample is a biological fluid sample or a biological tissue sample.
  • the biological fluid sample is a bodily fluid, such as blood, plasma, serum, cerebrospinal fluid (CSF) or brain interstitial fluid (ISF).
  • the biological sample is a biopsy sample.
  • the biological sample is a tissue or cell sample.
  • the biological sample is a B cell sample.
  • the biological sample is a sample enriched for certain molecules, such as immunoglobulin or target protein-binding molecules, for example by immunoprecipitation.
  • a plurality of sample is obtained timing the course of the treatment, e.g, every month, every 2 months, every three months, every 4 months, every 5 months, every 6 months, or every' year.
  • the sample for determining the level of one or more catabodies and the sample for determining the level of auto-antibodies or target protein are obtained from the individual at the same time, or are aliquots from the same sample.
  • the sample for determining the level of one or more catabodies and the sample for determining the level of auto-antibodies or target protein are obtained from the individual at different time and/or from different sources.
  • the same sample is used for determining the level of one or more SHD catabodies and the level of auto-antibodies against the target protein (e.g, Ab).
  • a serum sample is used for determining the level of one or more SHD catabodies
  • a CSF sample is used for determining the level of Ab.
  • the biological sample is a semm sample. In some embodiments, the biological sample is a semm sample.
  • the serum sample contains at least about 1 mg/mL, such as at least about any one of 2, 5, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, or more mg/mL lg.
  • the serum sample contains at least about 100 mg/mL Ig. In some embodiments, the serum sample contains no more than about any one of 500, 400, 300, 250, 200, or 150 mg/mL Ig.
  • the biological sample is incubated with the substrate peptide or the target protein (or fragment thereof, e.g., Ab) for about 1 hour to about 16 hours, including, for example, about any one of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 hours. In some embodiments, the biological sample is incubated with the substrate peptide or the target protein (or fragment thereof, e.g, Ab) for at least about any one of 1, 2, 3, 4, 5, 6,
  • the biological sample is incubated with the substrate peptide or the target protein (or fragment thereof, e.g. , Ab) for no more than about any one of 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 hour(s). In some embodiments, the biological sample is incubated with the substrate peptide or the target protein (or fragment thereof, e.g, Ab) for about 1 hour to about 3 hours. In some
  • the biological sample is incubated with the substrate peptide or the target protein (or fragment thereof, e.g, Ab) overnight.
  • the incubation is carried out at room temperature. In some embodiments, the incubation is carried out at 4°C.
  • the solid support e.g, ELISA plate
  • a wash buffer such as PBS or PBST.
  • the solid support e.g, ELISA plate
  • a blocking buffer such as 10% fetal bovine serum (FBS) in PBS, or 1% BSA in PBS.
  • the solid support e.g, ELISA plate
  • the solid support is washed (e.g, three times) after the blocking using a wash buffer, such as PBS or PBST.
  • the amount of the catabody-substrate peptide complex or the auto-antibody-target protein complex can be detected using an antibody that specifically binds to species-specific immunoglobulin molecules, such as human Ig.
  • the antibody specifically binds to total IgM, total lgG, total IgA, and/or total IgE.
  • Exemplary antibodies specifically binding to human Ig include, but are not limited to, goat anti-human Ig.
  • the antibody that specifically binds to Ig may be labeled with an enzyme (e.g., HRP) for detection using enhanced chemo-luminescence (ECL) substrates.
  • an enzyme e.g., HRP
  • the antibody that specifically binds to Ig may be labeled with a fluorescent label, e.g., FITC, for direct detection.
  • a plate reader may be used to detect the ECL signal or the fluorescence signal using suitable excitation, emission and cutoff wavelength settings.
  • the level of one or more auto-antibodies against die target protein is determined using an ELISA assay.
  • the level of the target protein (e.g, Ab) in a biological sample can be determined using an ELISA assay or a liquid
  • the level of one or more SHD catabodies is compared to a control SHD catabody level. In some embodiments, the level of one or more SHD catabodies is compared to the level of one or more SHD catabodies in a control sample. In some embodiments, the level of one or more SHD catabodies is compared to die level of one or more SHD catabodies in a plurality of control samples. In some embodiments, the plurality of control samples is used to generate a statistical distribution that is used to classify or rank the levels of one or more SHD catabodies in individuals of the same age or the same age group.
  • the level of an auto-antibody against a target protein is compared to a control auto-antibody level. In some embodiments, the level of an autoantibody against a target protein (e.g. , Ab) is compared to the level of the auto-antibody against the target protein (e.g. , Ab) in a control sample. In some embodiments, the level of an auto-antibody against a target protein (e.g. , Ab) is compared to the level of the auto-antibody against the target protein (e.g. , Ab) in a plurality of control samples.
  • the plurality of control samples are used to generate a statistical distribution that is used to classify or rank the levels of the auto-antibody against the target protein (e.g. , Ab) in a certain population of individuals, such as healthy individuals, individuals having a PAD (e.g, AD), or individuals of the same age or the same age group.
  • target protein e.g. , Ab
  • the level of the target protein is compared to a control target protein level. In some embodiments, the level of the target protein (e.g. , Ab) is compared to the level of the target protein (e.g, Ab) in a control sample. In some embodiments, the level of the target protein (e.g, Ab) is compared to the level of the target protein (e.g, Ab) in a plurality of control samples.
  • the plurality of control samples are used to generate a statistical distribution that is used to classify or rank the levels of the target protein (e.g, Ab) in a certain population of individuals, such as healthy individuals, individuals having a PAD (e.g, AD), or individuals of the same age or the same age group.
  • the target protein e.g, Ab
  • a certain population of individuals such as healthy individuals, individuals having a PAD (e.g, AD), or individuals of the same age or the same age group.
  • Exemplary age groups include, but are not limited to, 18-30 years old, 30-40 years old, 40-50 years old, 50-60 years old, 60-70 years old, 70-80 years old, 80-90 years old, 18- 40 years old, 40-90 years old, 18-60 years old, 60-90 years old, 60 years or older, 70 years or older, 75 years or older, 80 years or older, 85 years or older, or 90 years or older.
  • Control samples can be obtained using the same methods as non-control samples.
  • the control sample is obtained from a different individual, such as a healthy individual or an individual not having a PAD, and/or an individual sharing similar ethnic, age, and gender.
  • a plurality of control samples (for example from different individuals) is used to determine a range of levels of one or more SHD catabodies, auto-antibodies against the target protein (e.g. , Ab), or the target protein (e.g. ,
  • control SHD catabody level is the level of one or more SHD catabodies in a healthy individual.
  • control SHD catabody level is the average or median level of one or more SHD catabodies in a population of individuals, e.g., individuals of the same age group, or individuals of about 18 years old to about 50 years old (e.g., about 18 years old to about 40 years old, or about 18 years old to about 30 years old).
  • the control auto-antibody level is the level of the auto-antibody against the target protein (e.g, Ab) in a healthy individual.
  • control auto-antibody level is the median level of the auto-antibody against the target protein in a population of individuals, e.g, individuals of the same age group.
  • control target protein level is the level of the target protein (e.g. , Ab) in a healthy individual.
  • control target protein level is the median level of the target protein in a population of individuals, e.g. , individuals of the same age group.
  • the level of the one or more SHD catabodies, auto-antibodies against the target protein (e.g. , Ab), or the target protein (e.g. , Ab) is compared to a control or reference (e.g, the median or average level for a population of individuals or level of a healthy individual).
  • the control level is a pre-determined threshold level. For example, if the level of one or more SHD catabodies for an individual is determined to be no more than about any one of 90%, 80%, 75%, 70%, 60%, 50%, 40%,
  • the level of the one or more SHD catabodies for an individual is determined to be more than about any one of 20%, 50%, 75%, 2x, 3x, 5x, lOx or more than the median level of a population of individuals in the same age group, that individual is determined to have a high level of the one or more SHD catabodies.
  • the level of one or more auto-antibodies against the target protein (e.g , Ab) for an individual is determined to be no more than about any one of 90%, 80%, 75%, 70%, 60%, 50%, 40%, 30%, 20%, 10% or lower of the median level of a population of individuals in the same age group, that individual is determined to have a low level of the one or more autoantibodies against the target protein (e.g, Ab).
  • the level of the one or more auto-antibodies against the target protein (e.g, Ab) for an individual is determined to be more than about any one of 20%, 50%, 75%, 2x, 3x, 5x, lOx or more than the median level of a population of individuals in the same age group, that individual is determined to have a high level of the one or more auto-antibodies against the target protein (e.g , Ab).
  • the level of the target protein (e.g , Ab) for an individual is determined to be no more than about any one of 90%, 80%, 75%, 70%, 60%, 50%, 40%, 30%, 20%, 10% or lower of the median level of a population of individuals in the same age group, that individual is determined to have a low' level of the target protein (e.g, Ab).
  • the level of the target protein (e.g. , Ab) for an individual is determined to be more than about any one of 20%, 50%, 75%, 2x, 3x, 5x, lOx or more than the median level of a population of individuals in the same age group, that individual is determined to have a high level of the target protein (e.g., Ab).
  • an individual is determined as having a risk for a PAD (e.g, AD) if: (i) the level of the one or more SHD catabodies is lower than a control SHD catabody level; and (ii) the level of the auto-antibody against the target protein (e.g, Ab) is lower than a control auto-antibody level.
  • the level of the one or more SHD catabodies in an individual having a risk for a PAD is no more than about any one of 90%, 80%, 75%, 70%, 60%, 50%, 40%, 30%, 20%, 10% or lower of the median of a population of individuals in the same age group.
  • the level of the autoantibody against the target protein (e.g. , Ab) in an individual having a risk for a PAD is no more than about any one of 90%, 80%, 75%, 70%, 60%, 50%, 40%, 30%, 20%, 10% or lower of the median of a population of individuals in the same age group.
  • an individual is determined as having a risk for a PAD (e.g, AD) if: (i) the level of the one or more SHD catabodies is lower than a control SHD catabody level; and (ii) the level of tire target protein (e.g, Ab) is higher than a control target protein level.
  • the level of the one or more SHD catabodies in an individual having a risk for a PAD is no more than about any one of 90%, 80%, 75%, 70%, 60%, 50%, 40%, 30%, 20%, 10% or lower of the median of a population of individuals in the same age group.
  • the level of the target protein (e.g, Ab) in an individual having a risk for a PAD is more than about any one of 20%, 50%, 75%, 2x, 3x, 5x, lOx or more of the median of a population of individuals in the same age group.
  • the individual is a mammal and includes, but is not limited to, human, bovine, horse, feline, canine, rodent (mouse, rat or hamster), or non-human primate.
  • the individual is a human.
  • the individual is a young human individual, such as a human individual no more than about any one of 60, 50, 40, 30, or 25 years old.
  • the individual is an old human individual, such as a human individual older than about any one of 50, 60, 70, or 80 years old.
  • an“at risk” individual is an individual who is at risk of developing a PAD (e.g., AD).
  • An individual“at risk” may or may not have detectable disease, and may or may not have displayed detectable disease prior to the treatment methods described herein.
  • “At risk” denotes that an individual has one or more so-called risk factors, which are measurable parameters that correlate with development of a PAD (e.g., AD), which are described herein. An individual having one or more of these risk factors has a higher probability of developing a PAD (e.g, AD) than an individual without these risk factor(s).
  • PADs are known in the art and can be diagnosed, treated or prevented with the methods described herein.
  • Exemplary PADs include, but are not limited to, Alzheimer’s disease associated with accumulation of Ab; Parkinson’s disease associated with
  • a-synuclein Alzheimer’s disease or dementia associated with accumulation of Tau
  • ATTR amyloidosis associated with accumulation of transthyretin
  • AL amyloidosis associated with accumulation of immunoglobulin light chain
  • ubiquitin-positive neuronal and glial inclusions such as FTLD or ALS
  • TDP43 TAR DNA- binding protein of 43 kDa
  • Huntington’s disease associated with accumulation of
  • An therapeutic catabody that specifically bind to and cleave the target protein (e.g, Ab) can be administered to the individual if the individual is determined as having a PAD or having a risk of PAD, for example, if: (i) the level of the one or more SHD catabodies is lower than a control SHD catabody level; and (ii) the level of the auto-antibody against the target protein (e.g. , Ab) is lower than a control auto-antibody level, or the level of the target protein (e.g, Ab) is higher than a control target protein level.
  • the therapeutic catabody may be administered to the individual using any suitable dosage (including dosage amount and dosing schedule/fiequency) and routes of administration.
  • the dosage (or effective amount of the therapeutic catabody) may be determined according to the size and condition of the individual, and according to standard therapeutic practice.
  • the route of administration is in accordance with known and accepted methods, such as by single or multiple bolus or infusion over a period of time in a suitable manner, e.g. , injection or infusion by subcutaneous, intravenous, intraperitoneal, intramuscular, intra-arterial, intralesional, intraarticular, or oral routes. Animal experiments provide reliable guidance for the determination of effective doses for human diagnostic applications. Interspecies scaling of effective doses can be performed following the principles laid down by Mordenti, J. and Chappell, W.“The Use of Interspecies Scaling in Toxicokinetics,” In Toxicokinetics and New Drug Development, Yacobi etal., Eds, Peigamon Press, New York 1989, pp. 42-46.
  • the effective amount of a therapeutic catabody is about 1 pg/m 2 to about 100 mg/m 2 , or about 1 pg/kg to about 100 mg/kg.
  • the dosing frequency for the administration of the therapeutic catabody is from daily to about once every three months.
  • the administration of the therapeutic catabody can be extended over an extended period of time, such as from about a month up to years.
  • the level of one or more SHD catabodies, auto-antibodies against the target protein (e.g. , Ab) and/or the target protein (e.g. , Ab) is assessed
  • the level of one or more SHD catabodies, auto-antibodies against the target protein (e.g. , Ab) and/or the target protein (e.g. , Ab) is assessed about every month, every 2 months, every 3 months, every 4 months, every 6 months, or every year. In some
  • administration of the therapeutic catabody is repeated if: (i) the level of the one or more catabodies is lower than a control catabody level; and (ii) the level of the auto-antibody against the target protein (e.g., Ab) is lower than a control autoantibody level, or the level of the target protein (e.g. , Ab) is higher than a control target protein level.
  • the present application provides therapeutic catabodies that specifically bind and cleavage a target protein associated with a PAD.
  • the methods of treatment described in Section P may use any one of the therapeutic catabodies (e.g., anti-Ab catabodies) described in this section.
  • the therapeutic catabody cleaves a substrate having the formula EAR-AMC (SEQ ID NO: 1).
  • the therapeutic catabody is a catabody having an SHD motif in the light chain variable region (VL).
  • a catabody specifically bind and cleave amyloid beta (Ab) peptides.
  • the anti-Ab catabody cleaves a substrate having the formula EAR-AMC (SEQ ID NO: 1).
  • the anti-Ab catabody comprises an SHD motif in the light chain variable region (VL).
  • an isolated anti-Ab catabody derived from 3D6 there is provided an isolated anti-Ab catabody derived from 3D6.
  • the VL of the anti-Ab catabody is derived from 3D6, wherein the amino acid at position 1 of the VL is an Asp (D), and wherein the numbering is according to the EU index of Kabat.
  • the amino acid residue at position 26 of the VL is Ser (S)
  • the amino acid residue at position 27D of the VL is D
  • the amino acid residue at position 28 of the VL is D or Gin (N)
  • the numbering is according to the EU index of Kabat.
  • the heavy chain variable region (VH) of the anti-Ab catabody is derived from 3D6.
  • the VH of the anti- Ab catabody is screened from a phage library with human germline VH sequences based on binding affinity to Ab.
  • the anti-Ab catabody specifically binds to Ab competitively with 3D6.
  • an isolated anti-Ab catabody comprising: a VL comprising one, two, or three LC-CDRs of 3D6, wherein the amino acid residue at position 1 of the VL is D, the amino acid residue at position 27A of the VL is S, and the amino acid residue at position 93 of the VL is H, and wherein the numbering is according to the EU index of Kabat.
  • an isolated anti-Ab catabody comprising: a VL comprising a LC-CDR1, LC-CDR2, and LC-CDR3 of 3D6, wherein the amino acid residue at position 1 of the VL is D, the amino acid residue at position 27A of the VL is S, and the amino acid residue at position 93 of the VL is H, and wherein the numbering is according to the EU index of Kabat.
  • the amino acid residue at position 26 of the VL is Ser (S)
  • the amino acid residue at position 27D of the VL is D, E or H
  • the amino acid residue at position 28 of the VL is D or N, and wherein the numbering is according to the EU index of Kabat.
  • the anti-Ab catabody comprises a VH comprising one, two, or three HC-CDRs of 3D6. In some embodiments, the anti-Ab catabody comprises HC-CDR1, HC-CDR2, and HC-CDR3 of 3D6. In some embodiments, the VH of the anti-Ab catabody is screened from a phage library with human germline VH sequences based on binding affinity to Ab.
  • an isolated anti-Ab catabody comprising: a VL comprising a LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 12, LC- CDR2 comprising the amino acid sequence of SEQ ID NO: 13, and an LC-CDR3 comprising the amino acid sequence of SEQ P) NO: 14, or a variant thereof comprising up to about 5 (e.g., 1, 2, 3, 4, or 5) ammo acid substitutions in the LC-CDRs, wherein the amino acid residue at position 1 of the VL is D, the amino acid residue at position 27A of the VL is S, and the amino acid residue at position 93 of the VL is H, and wherein the numbering is according to the EU index of Kabat.
  • the amino acid residue at position 26 of the VL is Ser (S)
  • the amino acid residue at position 27D of the VL is D, E or H
  • the amino acid residue at position 28 of the VL is D or N
  • the numbering is according to the EU index of Rabat.
  • the anti-Ab catabody comprises a VH comprising an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 9, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 10, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 11, or a variant thereof comprising up to about 5 (e.g. ,
  • the VH of the anti-Ab catabody is screened from a phage library with human germline VH sequences based on binding affinity to Ab.
  • an isolated anti-Ab catabody comprising: a VL comprising a LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 12, LC- CDR2 comprising the amino acid sequence of SEQ ID NO: 13, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 14, wherein the amino acid residue at position 1 of the VL is D, the amino acid residue at position 27A of the VL is S, and the amino acid residue at position 93 of the VL is H, and wherein the numbering is according to the EU index of Rabat.
  • the amino acid residue at position 26 of the VL is Ser (S)
  • the amino acid residue at position 27D of the VL is D, E or H
  • the amino acid residue at position 28 of the VL is D or N
  • the numbering is according to the EU index of Rabat.
  • the anti-Ab catabody comprises a VH comprising an HC-CDR1 comprising the amino acid sequence of SEQ 1D NO: 9, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 10, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 11.
  • the VH of the anti-Ab catabody is screened from a phage library with human germline VH sequences based on binding affinity to Ab.
  • an isolated anti-Ab catabody comprising: a VL comprising a LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 12, LC- CDR2 comprising the amino acid sequence of SEQ ID NO: 13, and an LC-CDR3 comprising the amino acid sequence of SEQ P) NO: 14, wherein the amino acid residue at position 1 of the VL is D, the amino acid residue at position 27A of the VL is S, and the amino acid residue at position 93 of the VL is H, and wherein the numbering is according to the EU index of Rabat; and a VH comprising an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 9, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 10, and an HC- CDRS comprising the amino acid sequence of SEQ ID NO: 11.
  • the amino acid residue at position 26 of the VL is Ser (S)
  • the amino acid residue at position 27D of the VL is D, E or H
  • the amino acid residue at position 28 of the VL is D or N
  • the numbering is according to the EU index of Kabat.
  • an isolated anti-Ab catabody comprising: a VL comprising a LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 12, LC- CDR2 comprising the amino acid sequence of SEQ ID NO: 13, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 14, wherein the amino acid residue at position 1 of the VL is D, the amino acid residue at position 27A of the VL is S, and the amino acid residue at position 93 of the VL is H, the amino acid residue at position 26 of the VL is S, the amino acid residue at position 27D of the VL is D, E or H, and/or the amino acid residue at position 28 of the VL is D or N, and wherein the numbering is according to the EU index of Kabat; and a VH comprising an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 9, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 10, and an LC-CDR3 comprising the amino acid sequence
  • an isolated anti-Ab catabody comprising: a VL comprising the amino acid sequences of SEQ ID NOs: 12, 13 and 14, wherein the amino acid residue at position 1 of the VL is D, the amino acid residue at position 27A of the VL is S, and the amino acid residue at position 93 of the VL is H, and wherein the numbering is according to the EU index of Kabat.
  • the anti-Ab catabody comprises a VH comprising the amino acid sequences of SEQ ID NOs: 9, 10 and 11.
  • the amino acid residue at position 26 of the VL is Ser (S)
  • the amino acid residue at position 27D of the VL is D, E or H
  • the amino acid residue at position 28 of the VL is D or N
  • the numbering is according to the EU index of Kabat.
  • the VH of the anti-Ab catabody is screened from a phage library with human germline VH sequences based on binding affinity to Ab.
  • an isolated anti-Ab catabody comprising: a VL comprising an amino acid sequence having at least about 85% (e.g., at least about any one of 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to the amino acid sequence of SEQ ID NO: 5, 7 or 8, wherein the amino acid residue at position 1 of the VL is D, the amino acid residue at position 27A of the VL is S, and the amino acid residue at position 93 of the VL is H, and wherein the numbering is according to the EU index of Kabat.
  • the amino acid residue at position 26 of the VL is Ser (S)
  • the amino acid residue at position 27D of the VL is D, E or H
  • the amino acid residue at position 28 of the VL is D or N
  • the numbering is according to the EU index of Kabat.
  • the anti-Ab catabody comprises a VH comprising an amino acid sequence having at least about 85% (e.g ., at least about any one of 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to the amino acid sequence of SEQ ID NO: 4 or 6.
  • the VH of the anti-Ab catabody is screened from a phage library with human germline VH sequences based on binding affinity to Ab.
  • an isolated anti-Ab catabody comprising a VH comprising the amino acid sequence of SEQ ID NO: 4, and a VL comprising the amino acid sequence of SEQ ID NO: 5.
  • an isolated anti-Ab catabody comprising a VH comprising the amino acid sequence of SEQ ID NO: 6, and a VL comprising the amino acid sequence of SEQ ID NO: 7.
  • an isolated anti-Ab catabody comprising a VH comprising the amino acid sequence of SEQ ID NO: 6, and a VL comprising the amino acid sequence of SEQ ID NO: 8.
  • an isolated anti-Ab catabody comprising a VH comprising the amino acid sequence of SEQ ID NO: 6, and a VL comprising the amino acid sequence of SEQ ID NO: 21. In some embodiments, there is provided an isolated anti-Ab catabody comprising a VH comprising the amino acid sequence of SEQ ID NO: 6, and a VL comprising the amino acid sequence of SEQ ID NO: 22. In some embodiments, there is provided an isolated anti-Ab catabody comprising a VH comprising the amino acid sequence of SEQ ID NO: 19, and a VL comprising the amino acid sequence of SEQ ID NO: 7.
  • an isolated anti-Ab catabody comprising a VH comprising the amino acid sequence of SEQ ID NO: 19, and a VL comprising the amino acid sequence of SEQ ID NO: 8. In some embodiments, there is provided an isolated anti-Ab catabody comprising a VH comprising the amino acid sequence of SEQ ID NO: 19, and a VL
  • an isolated anti-Ab catabody comprising a VH comprising the amino acid sequence of SEQ P) NO: 19, and a VL comprising the amino acid sequence of SEQ P) NO: 22.
  • an isolated anti-Ab catabody comprising a VH comprising the amino acid sequence of SEQ ID NO: 20, and a VL comprising the amino acid sequence of SEQ ID NO: 7.
  • an isolated anti-Ab catabody comprising a VH comprising the amino acid sequence of SEQ ID NO: 20, and a VL comprising the amino acid sequence of SEQ ID NO: 8.
  • an isolated anti-Ab catabody comprising a VH comprising the amino acid sequence of SEQ ID NO: 20, and a VL comprising the amino acid sequence of SEQ ID NO: 21. In some embodiments, there is provided an isolated anti-Ab catabody comprising a VH
  • an isolated anti-Ab catabody that specifically binds to and cleaves Ab competitively with any one of the anti-Ab catabodies described herein.
  • the anti-Ab catabody is an antigen binding fragment, such as an scFv or a Fab.
  • the anti-Ab catabody comprises an antibody heavy chain constant region and an antibody light chain constant region.
  • the anti-Ab catabody is a full-length antibody, such as a full-length IgG antibody.
  • the full-length anti-Ab catabody is an IgA, IgD, IgE, IgG, or IgM antibody.
  • the full-length anti-Ab catabody comprises IgG constant domains, such as constant domains of any of IgGl, IgG2, IgG3, and IgG4 including variants thereof.
  • the anti-Ab catabody comprises an IgGl heavy chain constant region. In some embodiments, the anti-Ab catabody comprises an IgG2 heavy chain constant region. In some embodiments, the anti-Ab catabody comprises an IgG3 heavy chain constant region. In some embodiments, the anti-Ab catabody comprises an IgG4 heavy chain constant region. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 15. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 16. In some embodiments, the anti-Ab catabody comprises a kappa light chain constant region.
  • the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 17. In some embodiments, the anti-Ab catabody comprises a lambda light chain constant region. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 18. In some embodiments, the anti-Ab catabody comprises a kappa light chain constant region.
  • the anti-Ab catabody comprises an Fc region. In some embodiments, the anti-Ab catabody comprises an Fc region of a human IgG. In some embodiments, the anti-Ab catabody comprises an Fc region with enhanced antibody- dependent cellular cytotoxicity (ADCC) and/or complement dependent cytotoxicity (CDC) effector function. In some embodiments, the anti-Ab catabody comprises an Fc region with reduced ADCC and/or CDC effector function.
  • ADCC antibody- dependent cellular cytotoxicity
  • CDC complement dependent cytotoxicity
  • the anti-Ab catabody is murine, chimeric, humanized, or human.
  • a full-length anti-Ab catabody comprising IgGl constant domains
  • the anti-Ab catabody comprises: a VL comprising a LC- CDR1 comprising the amino acid sequence of SEQ ID NO: 12, LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 13, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 14, or a variant thereof comprising up to about 5 (e.g.
  • amino acid residues in the LC-CDRs wherein the amino acid residue at position 1 of the VL is D, the amino acid residue at position 27A of the VL is S, and the amino acid residue at position 93 of the VL is H, and wherein the numbering is according to the EU index of Rabat.
  • the amino acid residue at position 26 of the VL is Ser (S)
  • the amino acid residue at position 27D of the VL is D, E or H
  • amino acid residue at position 28 of the VL is D or N
  • the numbering is according to the EU index of Rabat.
  • the anti-Ab catabody comprises a VH comprising an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 9, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 10, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 11, or a variant thereof comprising up to about 5 (e.g., 1, 2, 3, 4, or 5) amino acid substitutions in the HC-CDRs.
  • the VH of the anti-Ab catabody is screened from a phage library with human germline VH sequences based on binding affinity to Ab.
  • the anti-Ab catabody comprises a VL comprising an amino acid sequence having at least about 85% sequence identity to the amino acid sequence of SEQ ID NO: 5, 7, 8, 21 or 22. In some embodiments, the anti-Ab catabody comprises a VH comprising an amino acid sequence having at least about 85% sequence identity to the amino acid sequence of SEQ ID NO: 4, 6, 19 or 20.
  • the anti-Ab catabody comprises: (i) a VH comprising the amino acid sequence of SEQ ID NO: 4, and a VL comprising the amino acid sequence of SEQ ID NO: 5; (ii) a VH comprising the amino acid sequence of SEQ ID NO: 6, and a VL comprising the amino acid sequence of SEQ ID NO: 7; (iii) a VH comprising the amino acid sequence of SEQ ID NO: 6, and a VL comprising the amino acid sequence of SEQ ID NO: 8; (iv) a VH comprising the amino acid sequence of SEQ ID NO: 19, and a VL comprising the amino acid sequence of SEQ ID NO: 21; (v) a VH comprising the amino acid sequence of SEQ ID NO: 20, and a VL comprising the amino acid sequence of SEQ ID NO: 21; (vi) a VH comprising the amino acid sequence of SEQ ID NO: 19, and a VL comprising the amino acid sequence of SEQ ID NO: 22; or (vii) a V
  • a full-length anti-Ab catabody comprising IgG4 constant domains
  • the anti-Ab catabody comprises: a VL comprising a LC- CDR1 comprising the amino acid sequence of SEQ ID NO: 12, LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 13, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 14, or a variant thereof comprising up to about 5 (e.g.
  • amino acid residues in the LC-CDRs wherein the amino acid residue at position 1 of the VL is D, the amino acid residue at position 27A of the VL is S, and the amino acid residue at position 93 of the VL is H, and wherein the numbering is according to the EU index of Kabat.
  • the amino acid residue at position 26 of the VL is Ser (S)
  • the amino acid residue at position 27D of the VL is D, E or H
  • amino acid residue at position 28 of the VL is D or N, and wherein the numbering is according to the EU index of Kabat.
  • the anti-Ab catabody comprises a VH comprising an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 9, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 10, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 11, or a variant thereof comprising up to about 5 (e.g., 1, 2, 3, 4, or 5) amino acid substitutions in the HC-CDRs.
  • the VH of the anti-Ab catabody is screened from a phage library with human germline VH sequences based on binding affinity to Ab.
  • the anti -A b catabody comprises a VL comprising an amino acid sequence having at least about 85% sequence identity to the amino acid sequence of SEQ ID NO: 5, 7, 8, 21 or 22. In some embodiments, the anti-Ab catabody comprises a VH comprising an amino acid sequence having at least about 85% sequence identity to the amino acid sequence of SEQ ID NO: 4, 6, 19 or 20.
  • the anti-Ab catabody comprises: (i) a VH comprising the amino acid sequence of SEQ ID NO: 4, and a VL comprising the amino acid sequence of SEQ ID NO: 5; (ii) a VH comprising the amino acid sequence of SEQ ID NO: 6, and a VL comprising the amino acid sequence of SEQ ID NO: 7; (iii) a VH comprising the amino acid sequence of SEQ ID NO: 6, and a VL comprising the amino acid sequence of SEQ ID NO: 8; (iv) a VH comprising the amino acid sequence of SEQ ID NO: 19, and a VL comprising the amino acid sequence of SEQ ID NO: 21 ; (v) a VH comprising the amino acid sequence of SEQ ID NO: 20, and a VL comprising the amino acid sequence of SEQ ID NO: 21; (vi) a VH comprising the amino acid sequence of SEQ ID NO: 19, and a VL comprising the amino acid sequence of SEQ ID NO: 22; or
  • catabodies described herein based on alternative prediction algorithms are within the scope of this invention.
  • catabodies comprising VH or VL sequences from catabodies described herein, but based on alternative algorithms are within the scope of this invention.
  • the anti-Ab catabody specifically binds to and cleaves an Ab peptide. In some embodiments, the anti-Ab catabody specifically binds to and cleaves a human Ab peptide, such as Ab(1-40) or Ab(1-42). In some embodiments, the anti-Ab catabody specifically binds to and cleaves an Ab peptide in helical conformation. In some embodiments, the anti-Ab catabody specifically binds to the N-terminus of Ab, such as N- terminal 3, 4, 5, 6, 7, 8, 9, 10, or more amino acids of Ab(1-40).
  • the anti-Ab catabody specifically binds to and cleaves an Ab oligomer. In some embodiments, the anti-Ab catabody specifically binds to and cleaves soluble Ab. In some embodiments, the anti-Ab catabody specifically binds to and cleaves Ab in amyloid plaques. In some embodiments, the anti-Ab catabody specifically binds to and cleaves Ab in the brain. In some embodiments, the anti-Ab catabody specifically binds to and cleaves vascular Ab.
  • the anti-Ab catabody cross-reacts with Ab from species other than human, such as mouse or rat. In some embodiments, the anti-Ab catabody is completely specific for human Ab and does not exhibit species or other types of non-human cross- reactivity. In some embodiments, the anti-Ab catabody cross-reacts with at least one allelic variant of Ab. In some embodiments, the anti-Ab catabody does not cross-react with any allelic variant of Ab.
  • Ab are peptides of 36-43 amino acids that have been implicated as the main component of amyloid plaques found in the brains of Alzheimer patients.
  • Ab peptides are derived from amyloid precursor protein (APP), which is cleaved by beta secretase and gamma secretase to yield Ab.
  • Ab molecules can aggregate to form flexible soluble oligomers which may exist in several forms and are toxic to neurons.
  • soluble oligomeric forms of the peptide may be causative agents in the development of Alzheimer's disease.
  • the Ab plaques are responsible for the pathology of Alzheimer's disease.
  • Brain Ab is elevated in patients with sporadic Alzheimer's disease.
  • Ab is the main constituent of brain parenchymal and vascular amyloid and it contributes to cerebrovascular lesions and is neurotoxic.
  • Ab circulates in plasma, cerebrospinal fluid (CSF) and brain interstitial fluid (ISF) mainly as soluble Ab40.
  • CSF cerebrospinal fluid
  • ISF brain interstitial fluid
  • Senile amyloid plaques contain both Ab40 and Ab42, while vascular amyloid is predominantly the shorter Ab40.
  • Several sequences of Ab were found in both lesions.
  • Ab42 is the most amyloidogenic form of the peptide.
  • Ab42 is also referred to as Ab(1-42).
  • Ab40 is also referred to as Ab(1-40).
  • Bapineuzumab is a humanized form of murine monoclonal antibody 3D6, which targets the N-terminal 5 residues of Ab peptide in a helical conformation.
  • Bapineuzumab was the first antibody to be found to cause amyloid-related imaging abnormalities, including an accumulation of fluid in brain tissue in patients receiving high doses. No health risks were found in patients receiving either 0.5 or 1 mg of bapineuzumab.
  • the VH of 3D6 comprises the amino acid sequence of SEQ ID NO: 4.
  • the VL of 3D6 comprises the amino acid sequence of SEQ ID NO: 23.
  • the VH of bapineuzumab comprises the amino acid sequence of SEQ ID NO: 24.
  • the VL of bapineuzumab comprises the amino acid sequence of SEQ ID NO: 25.
  • amino acid sequence variants of the therapeutic catabodies are contemplated.
  • Amino acid sequence variants of a catabody may be prepared by introducing appropriate modifications into the nucleotide sequence encoding the catabody, or by peptide synthesis. Such modifications include, for example, deletions from, and/or insertions into and/or substitutions of residues within the amino acid sequences of the catabody. Any combination of deletion, insertion, and substitution can be made to arrive at the final construct, provided that the final construct possesses the desired characteristics, e.g., antigen-binding and cleavage.
  • the catabody variants having one or more amino acid substitutions are provided.
  • Sites of interest for substitutional mutagenesis include the CDRs and FRs.
  • Amino acid substitutions may be introduced into a catabody of interest and the products screened for a desired activity, e.g., retained/improved antigen binding and cleavage, decreased immunogenicity, or improved ADCC or CDC.
  • Amino acids may be grouped into different classes according to common side-chain properties:
  • Non-conservative substitutions will entail exchanging a member of one of these classes for another class.
  • An exemplary substitutional variant is an affinity matured catabody, which may be conveniently generated, e.g., using phage display-based affinity maturation techniques. Briefly, one or more CDR residues are mutated and the variant catabodies displayed on phage and screened for a particular biological activity (e.g., binding affinity). Alterations (e.g, substitutions) may be made in HVRs, e.g, to improve antibody affinity. Such alterations may be made in HVR“hotspots,” i.e., residues encoded by codons that undergo mutation at high frequency during the somatic maturation process (see, e.g, Chowdhury, Methods Mol. Biol.
  • variable genes chosen for maturation are introduced into the variable genes chosen for maturation by any of a variety of methods (e.g, error-prone PCR, chain shuffling, or oligonucleotide-directed mutagenesis).
  • a secondary' library is then created. The library is then screened to identify any antibody variants with the desired affinity.
  • Another method to introduce diversity involves HVR-directed approaches, in which several HVR residues (e.g, 4-6 residues at a time) are randomized. HVR residues involved in antigen binding may be specifically identified, e.g, using alanine scanning mutagenesis or modeling. CDR-H3 and CDR-L3 in particular are often targeted.
  • substitutions, insertions, or deletions may occur within one or more CDRs so long as such alterations do not substantially reduce the ability of the catabody to bind and cleave antigen.
  • conservative alterations e.g, conservative substitutions as provided herein
  • binding affinity or antigen- cleavage activity may be made in CDRs.
  • Such alterations may be outside of HVR“hotspots” or SDRs.
  • each CDR either is unaltered, or contains no more than one, two or three amino acid substitutions.
  • a useful method for identification of residues or regions of an antibody that may be targeted for mutagenesis is called“alanine scanning mutagenesis” as described by
  • a residue or group of target residues e.g, charged residues such as Arg, Asp, His, Lys, and Glu
  • a neutral or negatively charged amino acid e.g, alanine or polyalanine
  • Further substitutions may be introduced at the ammo acid locations demonstrating functional sensitivity to the initial substitutions.
  • a crystal structure of an antigen-antibody complex can be determined to identify contact points between the antibody and antigen. Such contact residues and neighboring residues may be targeted or eliminated as candidates for substitution. Variants may be screened to determine whether they contain the desired properties.
  • the catabody variants described herein maintain their catalytic activity against the target protein (e.g, Ab).
  • the catalytic triad i.e. the SHD motif in the VL of the therapeutic catabody cannot be substituted.
  • one or more amino acid residues in the VL of the therapeutic catabody that support its catalytic activity cannot be substituted, including, for example, the amino acid residue at positions 26 , 27D, and 28 of the VL, wherein tire numbering is according to the EU index of Rabat.
  • Amino acid sequence insertions include amino- and/or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing a hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues.
  • terminal insertions include a catabody with an N-terminal methionyl residue.
  • Other insertional variants of the catabody include the fusion to the N- or C-terminus of the antibody to an enzyme (e.g. for ADEPT) or a polypeptide which increases the semm half-life of the antibody.
  • Catabody variants are also provided with amino-terminal leader extensions.
  • one or more amino acid residues of the amino-terminal leader sequence are present at the amino-terminus of any one or more heavy or light chains of an antibody.
  • An exemplary amino-terminal leader extension comprises or consists of three amino acid residues, VHS, present on one or both light chains of an antibody variant.
  • the therapeutic catabody provided herein is a chimeric antibody.
  • Certain chimeric antibodies are described, e.g., in U.S. Patent No. 4,816,567; and Morrison etal., (1984) Proc. Natl. Acad. Sci. USA, 81 :6851-6855 (1984)).
  • a chimeric antibody comprises anon-human variable region (e.g., a variable region derived from a mouse) and a human constant region.
  • a chimeric antibody is a “class switched” antibody in which the class or subclass has been changed from that of the parent antibody. Chimeric antibodies include antigen-binding fragments thereof.
  • humanized catabodies are provided.
  • Humanized antibodies are useful as therapeutic molecules because humanized antibodies reduce or eliminate the human immune response to non-human antibodies (such as the human anti-mouse antibody (HAMA) response), which can result in an immune response to an antibody therapeutic, and decreased effectiveness of the therapeutic.
  • HAMA human anti-mouse antibody
  • a chimeric antibody is a humanized antibody.
  • a non-human antibody is humanized to reduce immunogenicity to humans, while retaining the specificity and affinity' of the parental non-human antibody.
  • a humanized antibody comprises one or more variable domains in which HVRs, e.g., CDRs, (or portions thereof) are derived from a non-human antibody, and FRs (or portions thereof) are derived from human antibody sequences.
  • HVRs e.g., CDRs, (or portions thereof) are derived from a non-human antibody
  • FRs or portions thereof
  • a humanized antibody optionally will also comprise at least a portion of a human constant region.
  • some FR residues in a humanized antibody are substituted with corresponding residues from anon-human antibody (e.g., the antibody from which the HVR residues are derived), e.g, to restore or improve antibody specificity or affinity.
  • anon-human antibody e.g., the antibody from which the HVR residues are derived
  • Human framework regions that may be used for humanization include but are not limited to: ftamework regions selected using the“best-fit” method (see, e.g, Sims etal. (1993) J. Immunol. 151 :2296); framework regions derived from the consensus sequence of human antibodies of a particular subgroup of light or heavy chain variable regions (see, e.g, Carter et al. (1992) Proc. Natl. Acad. Sci. USA, 89:4285; and Presta et al. (1993) J. Immunol, 151 :2623); human mature (somatically mutated) framework regions or human germline framework regions (see, e.g., Almagro and Fransson, (2008) Front.
  • the therapeutic catabodies may be isolated by screening combinatorial libraries for antibodies with the desired activity or activities. For example, a variety of methods are known in the art for generating phage display libraries and screening such libraries for antibodies possessing the desired binding characteristics. Such methods are reviewed, e.g., in
  • phage display methods repertoires of VH and VL genes are separately cloned by polymerase chain reaction (PCR) and recombined randomly in phage libraries, which can then be screened for antigen-binding phage as described in Winter et al., Ann Rev. Immunol., 12: 433-455 (1994). Phage typically displays antibody fragments, either as scFv fragments or as Fab fragments. Libraries from immunized sources provide high-affinity antibodies to the immunogen without the requirement of constructing hybridomas.
  • PCR polymerase chain reaction
  • naive repertoire can be cloned (e.g., from human) to provide a single source of antibodies to a wide range of non-self and also self-antigens without any immunization as described by Griffiths et al., EMBOJ, 12: 725-734 (1993).
  • naive libraries can also be made synthetically by cloning unrearranged V-gene segments from stem cells, and using PCR primers containing random sequence to encode the highly variable CDR3 regions and to accomplish rearrangement in vitro, as described by Hoogenboom and Winter, J. Mol. Biol. , 227: 381-388 (1992).
  • Patent publications describing human antibody phage libraries include, for example: US Patent No. 5,750,373, and US Patent Publication Nos. 2005/0079574, 2005/0119455, 2005/0266000, 2007/0117126, 2007/0160598, 2007/0237764, 2007/0292936, and 2009/0002360.
  • one or more amino acid modifications may be introduced into the Fc region of the therapeutic catabodies (such as anti-Ab catabodies) provided herein, thereby generating an Fc region variant.
  • the Fc region variant has enhanced ADCC effector function, often related to binding to Fc receptors (FcRs).
  • FcRs Fc receptors
  • the Fc region variant has decreased ADCC effector function.
  • changes or mutations to Fc sequences that can alter effector function. For example, WO 00/42072 and Shields etal. JBiol. Chem. 9(2): 6591-6604 (2001) describe antibody variants with improved or diminished binding to FcRs. The contents of those publications are specifically incorporated herein by reference.
  • the therapeutic catabody comprises an Fc region that possesses some but not all effector functions, which makes it a desirable candidate for applications in which the half-life of the catabody in vivo is important yet certain effector functions (such as CDC and ADCC) are unnecessary or deleterious.
  • In vitro and/or in vivo cytotoxicity assays can be conducted to confirm the reduction/depletion of CDC and/or ADCC activities.
  • Fc receptor (FcR) binding assays can be conducted to ensure that the antibody lacks FcyR binding (hence likely lacking ADCC activity), but retains FcRn binding ability.
  • NK cells express FcyRIII only, whereas monocytes express FcyRI, FcyRII and FcyRIII.
  • FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991).
  • Nonlimiting examples of in vitro assays to assess ADCC activity of a molecule of interest is described in U.S. Pat. No. 5,500,362 (see, e.g. Hellstrom, I. etal. Proc. Natl Acad. Sci. USA 83:7059-7063 (1986)) and Hellstrom, I etal., Proc. Nat'lAcad.
  • non-radioactive assay methods may be employed (see, for example, ACTTTM non-radioactive cytotoxicity assay for flow cytometry (CellTechnology, Inc.
  • PBMC peripheral blood mononuclear cells
  • NK Natural Killer
  • ADCC activity of the molecule of interest may be assessed in vivo, e.g. , in an animal model such as that disclosed in Clynes etal. Proc. Nat'lAcad. Sci. USA 95:652-656 (1998).
  • Clq binding assays may also be carried out to confirm that the antibody is unable to bind C 1 q and hence lacks CDC activity. See, e.g., Clq and C3c binding ELISA in WO 2006/029879 and WO
  • a CDC assay may be performed (see, for example, Gazzano-Santoro etal., J. Immunol. Methods 202: 163 (1996); Cragg, M. S. etal, Blood 101 : 1045-1052 (2003); and Cragg, M. S. and M. J. Glennie, Blood 103:2738-2743 (2004)).
  • FcRn binding and in vivo clearance/half-life determinations can also be performed using methods known in the art (see, e.g, Petkova, S. B. etal, Inti Immunol. 18(12): 1759- 1769 (2006)).
  • Antibodies with reduced effector function include those with substitution of one or more of Fc region residues 238, 265, 269, 270, 297, 327 and 329 (U.S. Pat. No. 6,737,056).
  • Fc mutants include Fc mutants with substitutions at two or more of amino acid positions 265, 269, 270, 297 and 327, including the so-called“DANA” Fc mutant with substitution of residues 265 and 297 to alanine (U.S. Pat. No. 7,332,581).
  • Antibodies with increased half-lives and improved binding to FcRn are described in US2005/0014934A1 (Hinton et ai). Those antibodies comprise an Fc region with one or more substitutions therein which improve binding of the Fc region to FcRn.
  • Fc variants include those with substitutions at one or more of Fc region residues: 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424 or 434, e.g, substitution of Fc region residue 434 (U.S. Pat. No. 7,371,826).
  • the therapeutic catabody (such as anti-Ab catabody) provided herein is altered to increase or decrease the extent to which tire construct is glycosylated.
  • Addition or deletion of glycosylation sites to an antibody may be conveniently accomplished by altering the amino acid sequence of the antibody such that one or more glycosylation sites is created or removed.
  • the carbohydrate attached thereto may be altered.
  • Native antibodies produced by mammalian cells typically comprise a branched, biantennary oligosaccharide that is generally attached by an N-linkage to Asn297 of the CH2 domain of the Fc region. See, e.g., Wright etal, TIBTECH 15:26-32 (1997).
  • the oligosaccharide may include various carbohydrates, e.g., mannose, N-acetyl glucosamine (GlcNAc), galactose, and sialic acid, as well as a fucose attached to a GlcNAc in the“stem’' of the biantennary oligosaccharide structure.
  • modifications of the oligosaccharide in the catabody may be made in order to create catabody variants with certain improved properties.
  • N-glycans attached to the CH2 domain of Fc is heterogeneous.
  • Antibodies or Fc fusion proteins generated in CHO cells are fucosylated by fucosyltransferase activity. See Shoji-Hosaka etal., J. Biochem. 2006, 140:777- 83. Normally, a small percentage of naturally occurring afucosylated IgGs may be detected in human serum.
  • N-glycosylation of the Fc is important for binding to FcyR; and afucosylation of the N-glycan increases Fc's binding capacity to FcyRIlla. Increased FcyRIIIa binding can enhance ADCC, which can be advantageous in certain therapeutic applications in which cytotoxicity is desirable.
  • an enhanced effector function can be detrimental when remediated cytotoxicity is undesirable.
  • the Fc fragment or CH2 domain is not glycosylated.
  • the N-glycosylation site in the CFG domain is mutated to prevent from glycosylation.
  • catabody variants having a carbohydrate structure that lacks fucose attached (directly or indirectly) to an Fc region.
  • the amount of fucose in such antibody may be from 1% to 80%, from 1% to 65%, from 5% to 65% or from 20% to 40%.
  • the amount of fucose is determined by calculating the average amount of fucose within the sugar chain at Asn297, relative to the sum of all glycostructures attached to Asn 297 (e.g., complex, hybrid and high mannose structures) as measured by MALDI-TOF mass spectrometry, as described in WO 2008/077546, for example.
  • Asn297 refers to the asparagine residue located at about position 297 in the Fc region (EU numbering of Fc region residues); however, Asn297 may also be located about ⁇ 3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300, due to minor sequence variations in antibodies. Such fucosylation variants may have improved ADCC function. See, e.g., US Patent Publication Nos. US 2003/0157108 (Presta, L.); US 2004/0093621 (Kyowa Hakko Kogyo Co., Ltd).
  • Examples of publications related to“defucosylated” or“fiicose- deficient” antibody variants include: US 2003/0157108; WO 2000/61739; WO 2001/29246; US 2003/0115614; US 2002/0164328; US 2004/0093621; US 2004/0132140; US
  • Catabody variants are further provided with bisected oligosaccharides, e.g., in which a biantennary oligosaccharide attached to the Fc region of the antibody is bisected by GlcNAc.
  • Such antibody variants may have reduced fucosylation and/or improved ADCC function. Examples of such antibody variants are described, e.g., in WO 2003/011878 (Jean-Mairet et al.); US Patent No. 6,602,684 (Umana etal); and US 2005/0123546 (Umanaeto/.).
  • Antibody variants with at least one galactose residue in the oligosaccharide attached to the Fc region are also provided. Such antibody variants may have improved CDC function. Such antibody variants are described, e.g., in WO 1997/30087 (Patel etal.); WO 1998/58964 (Raju, S.); and WO 1999/22764 (Raju, S.).
  • the therapeutic catabodies (such as anti-Ab catabodies) provided herein may be further modified to contain additional non-proteinaceous moieties that are known in the art and readily available.
  • the moieties suitable for derivatization of the catabodies include but are not limited to water soluble polymers.
  • Non-limiting examples of water soluble polymers include, but are not limited to, polyethylene glycol (PEG), copolymers of ethylene glycol/propylene glycol, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly-1, 3-dioxolane, poly-l,3,6-trioxane, ethylene/maleic anhydride copolymer, polyaminoacids (either homopolymers or random copolymers), and dextran or poly(n-vinyl pyrrolidone)polyethylene glycol, propropylene glycol homopolymers, prolypropylene oxide/ethylene oxide co-polymers, polyoxyethylated polyols (e g., glycerol), polyvinyl alcohol, and mixtures thereof.
  • PEG polyethylene glycol
  • copolymers of ethylene glycol/propylene glycol carboxymethylcellulose
  • dextran polyvinyl alcohol
  • Polyethylene glycol propionaldehyde may have advantages in manufacturing due to its stability in water.
  • the polymer may be of any molecular weight, and may be branched or unbranched.
  • the number of polymers attached to the catabody may vary, and if more than one polymer are attached, they can be the same or different molecules. In general, the number and/or type of polymers used for derivatization can be determined based on considerations including, but not limited to, the particular properties or functions of the catabody to be improved, whether the catabody derivative will be used in a therapy under defined conditions, etc.
  • the therapeutic catabodies (such as anti-Ab catabodies) described herein can be prepared using any known methods in the art, including those described below and in the Examples.
  • Therapeutic catabodies can be obtained by immunizing lab animals against target antigens, such as transition-state analogs. See, for example, US2010018361A1 and Taguchi H, et al.“Catalytic antibodies to amyloid beta peptide in defense against Alzheimer disease,” Autoimmun. Rev. 7:391-397 (2008), which are incorporated herein by reference. Therapeutic catabodies may also be expressed recombinantly.
  • nucleic acid molecules comprising polynucleotides that encode one or more chains of the therapeutic catabodies (such as anti- Ab catabodies) described herein.
  • a nucleic acid molecule comprises a polynucleotide that encodes a heavy chain or a light chain of the therapeutic catabody (such as anti-Ab catabody).
  • a nucleic acid molecule comprises both a polynucleotide that encodes a heavy chain and a polynucleotide that encodes a light chain of the therapeutic catabody (such as anti-Ab catabody).
  • a first nucleic acid molecule comprises a first polynucleotide that encodes a heavy chain and a second nucleic acid molecule comprises a second polynucleotide that encodes a light chain.
  • the first polynucleotide that encodes the heavy chain is operably linked to a first promoter
  • the second polynucleotide that encodes the light chain is operably linked to a second promoter.
  • the polynucleotide that encodes the heavy chain and the polynucleotide that encodes the light chain is operably linked to a promoter.
  • Additional promoter elements e.g., enhancers, regulate the frequency of
  • transcriptional initiation typically, these are located in the region 30-110 bp upstream of the start site, although a number of promoters have recently been shown to contain functional elements downstream of the start site as well.
  • the spacing between promoter elements frequently is flexible, so that promoter function is preserved when elements are inverted or moved relative to one another.
  • tk thymidine kinase
  • a suitable promoter is the immediate early cytomegalovirus (CMV) promoter sequence.
  • CMV immediate early cytomegalovirus
  • This promoter sequence is a strong constitutive promoter sequence capable of driving high levels of expression of any polynucleotide sequence operatively linked thereto.
  • Another example of a suitable promoter is Elongation Growth Factor-la (EF- la).
  • constitutive promoter sequences may also be used, including, but not limited to the simian vims 40 (SV40) early promoter, mouse mammary tumor vims (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, an avian leukemia vims promoter, an Epstein-Barr vims immediate early promoter, a Rous sarcoma vims promoter, as well as human gene promoters such as, but not limited to, the actin promoter, the myosin promoter, the hemoglobin promoter, and the creatine kinase promoter. Further, the invention should not be limited to the use of constitutive promoters.
  • inducible promoters are also contemplated as part of the invention.
  • the use of an inducible promoter provides a molecular switch capable of turning on expression of the polynucleotide sequence which it is operatively linked when such expression is desired, or turning off the expression when expression is not desired.
  • inducible promoters include, but are not limited to a metallothionine promoter, a glucocorticoid promoter, a progesterone promoter, and a tetracycline promoter.
  • the expression of the therapeutic catabody is inducible.
  • a nucleic acid sequence encoding the therapeutic catabody is operably linked to an inducible promoter.
  • a polynucleotide encoding a heavy chain or right chain of therapeutic catabody comprises a nucleotide sequence that encodes a leader sequence, which, when translated, is located at the N terminus of the heavy- chain or light chain.
  • the leader sequence may be the native heavy or right chain leader sequence, or may be another heterologous leader sequence.
  • the nucleic acid (or a set of nucleic acids) encoding the therapeutic catabody (such as anti-Ab catabody) may further comprises a nucleic acid sequence encoding a peptide tag (such as protein purification tag, e.g., His-tag, HA tag).
  • the present application also includes variants to these nucleic acid sequences.
  • the variants include nucleotide sequences that hybridize to the nucleic acid sequences encoding any one of the therapeutic catabodies described herein under at least moderately stringent hybridization conditions.
  • Nucleic acid molecules may be constructed using recombinant DNA techniques conventional in the art.
  • a nucleic acid molecule is an expression vector that is suitable for expression in a selected host cell.
  • Vectors comprising polynucleotides that encode the heavy chains and/or light chains of any one of the therapeutic catabodies (such as anti-Ab catabodies) described herein are provided.
  • Such vectors include, but are not limited to, DNA vectors, phage vectors, viral vectors, retroviral vectors, etc.
  • a vector comprises a first
  • the heavy chain and light chain are expressed from the vector as two separate polypeptides. In some embodiments, the heavy chain and light chain are expressed as part of a single polypeptide.
  • a first vector comprises a polynucleotide that encodes a heavy chain and a second vector comprises a polynucleotide that encodes a light chain.
  • the first vector and second vector are transfected into host cells in similar amounts (such as similar molar amounts or similar mass amounts).
  • a mole- or mass-ratio of between 5: 1 and 1:5 of the first vector and the second vector is transfected into host cells.
  • a mass ratio of between 1: 1 and 1:5 for the vector encoding the heavy- chain and the vector encoding the light chain is used.
  • a mass ratio of 1 :2 for the vector encoding the heavy chain and the vector encoding the light chain is used.
  • the nucleic acid can be cloned into a number of types of vectors.
  • the nucleic acid can be cloned into a vector including, but not limited to a plasmid, a phagemid, a phage derivative, an animal virus, and a cosmid.
  • Vectors of particular interest include expression vectors, replication vectors, probe generation vectors, and sequencing vectors.
  • the expression vector may be provided to a cell in the form of a viral vector.
  • Viral vector technology is well known in the art and is described, for example, in Green and Sambrook (2013, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York), and in other virology and molecular biology manuals.
  • Viruses which are useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentivirases.
  • a suitable vector contains an origin of replication functional in at least one organism, a promoter sequence, convenient restriction endonuclease sites, and one or more selectable markers (see, e.g., WO 01/96584; WO 01/29058; and U.S. Pat. No. 6,326,193).
  • the expression of the therapeutic catabody by a natural or synthetic nucleic acid encoding the catabody can be achieved by inserting the nucleic acid into an appropriate expression vector, such that the nucleic acid is operably linked to 5’ and 3’ regulatory elements, including for example a promoter (e.g. , a lymphocyte-specific promoter) and a 3’ untranslated region (UTR).
  • the vectors can be suitable for replication and integration in eukaryotic host cells.
  • Typical cloning and expression vectors contain transcription and translation terminators, initiation sequences, and promoters useful for regulation of the expression of the desired nucleic acid sequence.
  • a vector is selected that is optimized for expression of polypeptides in CHO or CHO-derived cells, or in NSO cells. Exemplary such vectors are described, e.g., in Running Deer etal., Biotechnol. Prog. 20:880-889 (2004).
  • the expression vector to be introduced into a cell can also contain either a selectable marker gene or a reporter gene or both to facilitate identification and selection of expressing cells from the population of cells sought to be transfected or infected through viral vectors.
  • the selectable marker may be carried on a separate piece of DNA and used in a co- transfection procedure. Both selectable markets and reporter genes may be flanked with appropriate regulatory sequences to enable expression in the host cells.
  • Useful selectable markers include, for example, antibiotic-resistance genes, such as neo and the like.
  • Reporter genes are used for identifying potentially transfected cells and for evaluating the functionality of regulatory sequences.
  • a reporter gene is a gene that is not present in or expressed by the recipient organism or tissue and that encodes a polypeptide whose expression is manifested by some easily detectable property, e.g., enzymatic activity. Expression of the reporter gene is assayed at a suitable time after the DNA has been introduced into the recipient cells.
  • Suitable reporter genes may include genes encoding luciferase, b-galactosidase, chloramphenicol acetyl transferase, secreted alkaline phosphatase, or the green fluorescent protein gene (e.g., Ui-Tel etal., 2000 FEBS Letters 479: 79-82). Suitable expression systems are well known and may be prepared using known techniques or obtained commercially. In general, the construct with the minimal 5’ flanking region showing the highest level of expression of reporter gene is identified as the promoter. Such promoter regions may be linked to a reporter gene and used to evaluate agents for the ability to modulate promoter-driven transcription.
  • the present application provides isolated host cells comprising any one of the therapeutic catabodies (such as anti-Ab catabodies), nucleic acid molecules, or vectors described herein.
  • the therapeutic catabodies (such as anti-Ab catabodies) described herein may be expressed in prokaryotic cells, such as bacterial cells; or in eukaryotic cells, such as fungal cells (such as yeast), plant cells, insect cells, and mammalian cells. Such expression may be carried out, for example, according to procedures known in the art.
  • exemplary eukaryotic cells that may be used to express polypeptides include, but are not limited to, COS cells, including COS 7 cells; 293 cells, including 293-6E cells; CHO cells, including CHO-S, DG44. Lecl3 CHO cells, and FUT8 CHO cells; PER.C6 ® cells (Crucell); and NSO cells.
  • Suitable non-mammalian host cells include prokaryotes (such as E. coli or B. subtillis) and yeast (such as S. cerevisae, S. pombe; or K lactis).
  • a particular eukaryotic host cell is selected based on its ability to make desired post-translational modifications to the heavy chains and/or light chains of the antibody.
  • CHO cells produce polypeptides that have a higher level of sialylation than the same polypeptide produced in 293 cells.
  • Nucleic acids may be transiently or stably transfected in the desired host cells, according to any suitable method.
  • the therapeutic catabody is produced in a cell-free system.
  • a cell-free system Non-limiting exemplary cell-free systems are described, e.g, in Sitaraman et al., Methods Mol. Biol. 498: 229-44 (2009); Spirin, Trends Biotechnol. 22: 538-45 (2004); Endo etal, Biotechnol. Adv. 21: 695-713 (2003).
  • the therapeutic catabodies may be purified by any suitable method. Such methods include, but are not limited to, the use of affinity matrices or hydrophobic interaction chromatography.
  • Suitable affinity ligands include ligands that bind antibody constant regions.
  • a Protein A, Protein G, Protein A/G, or an antibody affinity column may be used to bind the constant region and to purify an antibody comprising an Fc fragment.
  • Hydrophobic interactive chromatography for example, a butyl or phenyl column, may also suitable for purifying some polypeptides such as antibodies. Ion exchange chromatography (e.g. anion exchange chromatography and/or cation exchange
  • chromatography may also suitable for purifying some polypeptides such as antibodies.
  • Mixed-mode chromatography e.g. reversed phase/anion exchange, reversed phase/cation exchange, hydrophilic interaction/anion exchange, hydrophilic interaction/cation exchange, etc.
  • Many methods of purifying polypeptides are known in the art. V. Compositions, Kits and Articles of manufacture
  • compositions comprising any one of the therapeutic catabodies (such as anti-Ab catabodies), nucleic acids, vectors, or host cells described herein.
  • compositions of the therapeutic catabodies (such as anti-Ab catabodies) described herein can be obtained by mixing the therapeutic catabodies (such as anti-Ab catabodies) having the desired degree of purity with optional pharmaceutically acceptable carriers, excipients or stabilizers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)), in the form of lyophilized formulations or aqueous solutions.
  • Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as olyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine
  • Lyophilized formulations adapted for subcutaneous administration are described in W097/04801. Such lyophilized formulations may be reconstituted with a suitable diluent to a high protein concentration and the reconstituted formulation may be administered subcutaneously to the individual to be imaged, diagnosed, or treated herein.
  • compositions to be used for in vivo administration must be sterile.
  • kits useful for any one of the methods of determining catabody levels, diagnosis and treatment described herein including kits comprising any one of the therapeutic catabodies (such as anti-Ab catabodies) described herein.
  • kits for determining catabody levels in a biological sample comprising a substrate peptide comprises the amino acid sequence (EAR)n (SEQ ID NO: 2), and wherein n is an integer between 1 and 30 (e.g., n is 3).
  • the kit further comprises an antibody that specifically binds to total Ig (e.g., total human Ig, such as total IgM, total IgG, total IgA, and/or total IgE).
  • total Ig e.g., total human Ig, such as total IgM, total IgG, total IgA, and/or total IgE.
  • the kit comprises a solid support (e.g, ELISA plate).
  • a solid support e.g, ELISA plate
  • kits for diagnosing a PAD or determining a risk for a PAD in an individual, wherein the PAD is associated with a target protein comprising: a) a substrate peptide comprises the amino acid sequence (EAR)n (SEQ ID NO:
  • the kit further comprises an antibody that specifically binds to total Ig (e.g. , total human Ig, such as total IgM, total IgG, total IgA, and/or total IgE).
  • the kit comprises a solid support (e.g, ELISA plate).
  • kits for diagnosing AD or determining a risk for AD in an individual comprising: a) a substrate peptide comprises the amino acid sequence (EAR) n (SEQ ID NO: 2), and wherein n is an integer between 1 and 30 (e.g, n is
  • the kit comprises a solid support (e.g., ELISA plate).
  • kits for treating or preventing a PAD in an individual comprising: a) a substrate peptide comprises the amino acid sequence (EAR) n (SEQ ID NO: 2), and wherein n is an integer between 1 and 30 (e.g. , n is 3); b) the target protein (or a fragment thereof) or an antibody against the target protein; c) an antibody that specifically binds to total Ig (e.g. , total human Ig, such as total IgM, total IgG, total IgA, and/or total IgE); and d) a therapeutic catabody that specifically binds to the target protein.
  • the kit comprises a solid support (e.g, ELISA plate).
  • kits for treating or preventing AD in an individual comprising: a) a substrate peptide comprises the amino acid sequence (EAR)n (SEQ ID NO: 2), and wherein n is an integer between 1 and 30 (e.g, n is 3); b) an Ab peptide (e.g, Ab(1-42)) or an anti-Ab antibody; c) an antibody that specifically binds to total Ig (e.g, total human Ig, such as total IgM, total IgG, total IgA, and/or total IgE); and d) a therapeutic catabody that specifically binds to Ab, such as any one of the anti-Ab catabodies described herein.
  • a substrate peptide comprises the amino acid sequence (EAR)n (SEQ ID NO: 2), and wherein n is an integer between 1 and 30 (e.g, n is 3); b) an Ab peptide (e.g, Ab(1-42)) or an anti-Ab antibody; c) an antibody that specifically binds to total I
  • the kit comprises a solid support (e.g, ELISA plate).
  • a kit for treating or preventing AD in an individual comprising a pharmaceutical composition comprising an anti-Ab catabody and a pharmaceutically acceptable carrier, wherein the anti-Ab catabody comprises: a VL comprising a LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 12, LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 13, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 14, or a variant thereof comprising up to about 5 amino acid substitutions in the LC-CDRs, wherein the amino acid residue at position 1 of the VL is D, the amino acid residue at position 27A of the VL is S, and the amino acid residue at position 93 of the VL is H, and wherein the numbering is according to the EU index of Kabat.
  • the amino acid residue at position 26 of the VL is Ser (S)
  • the amino acid residue at position 27D of the VL is D, E or H
  • the amino acid residue at position 28 of the VL is D or N
  • the numbering is according to the EU index of Kabat.
  • the anti-Ab catabody comprises a VH comprising an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 9, an HC-CDR2 comprising tire amino acid sequence of SEQ ID NO: 10, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 11, or a variant thereof comprising up to about 5 amino acid substitutions in the HC-CDRs.
  • kits of the present application are in suitable packaging.
  • suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging (e.g., sealed Mylar or plastic bags), and the like.
  • Kits may optionally provide additional components such as reagents (e.g., ECL substrate), buffers (e.g, coating buffer, blocking buffer, washing buffer, antibody dilution buffer, developing buffer, etc.), antibodies (e.g, anti-human Ig antibody), and interpretative information.
  • reagents e.g., ECL substrate
  • buffers e.g, coating buffer, blocking buffer, washing buffer, antibody dilution buffer, developing buffer, etc.
  • antibodies e.g, anti-human Ig antibody
  • the present application thus also provides articles of manufacture.
  • the article of manufacture can comprise a container and a label or package insert on or associated with the container.
  • Suitable containers include vials (such as sealed vials), bottles, jars, flexible packaging, and the like.
  • the container holds a pharmaceutical composition, and may have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle).
  • the container holds substrate peptides or target protein (e.g, Ab) for use in the immunoassays.
  • the label or package insert indicates that the composition is used for diagnosing (including determining a risk), treating or preventing a PAD (e.g, AD) in an individual.
  • the label or package insert will further comprise instructions fir performing the immunoassays to determine one or more SHD catabody levels in a biological sample, and/or administering the pharmaceutical composition to the individual.
  • the label may indicate directions for reconstitution and/or use of the various components.
  • the container holding the pharmaceutical composition may be a multi-use vial, which allows for repeat administrations (e.g. from 2-6 administrations) of the reconstituted formulation.
  • Package insert refers to instructions customarily included in commercial packages of diagnostic and/or therapeutic products that contain information about the indications, usage, dosage, administration, contraindications and/or warnings concerning the use of such products.
  • the article of manufacture may further comprise a second container comprising a pharmaceutically-acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution and dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.
  • a pharmaceutically-acceptable buffer such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution and dextrose solution.
  • kits or article of manufacture may include multiple unit doses of the
  • compositions and instructions for use packaged in quantities sufficient for storage and use in pharmacies, for example, hospital pharmacies and compounding pharmacies.
  • Embodiments 1 A method for determining the level of one or more SHD catabodies in a biological sample, comprising:
  • the substrate peptide comprises the amino acid sequence (EAR) n (SEQ ID NO: 2), and wherein n is an integer between 1 and 30.
  • Embodiments 2 The method of embodiment 1, wherein n is 3.
  • Embodiments 3 The method of embodiment 1 or 2, wherein the biological sample is a serum sample.
  • Embodiments 4 The method of embodiment 3, wherein the serum sample contains at least about 1 mg/mL immunoglobulin (Ig).
  • Embodiments 5 The method of any one of embodiments 1-4, wherein the biological sample is incubated with the substrate peptide for about 1 hour to about 16 hours.
  • Embodiments 6. The method of any one of embodiments 1-5, wherein the amount of the catabody-substrate peptide complex is determined using an antibody that specifically binds to total Ig.
  • Embodiments 7 The method of embodiment 6, wherein the antibody is labeled with an enzyme or a fluorescent label.
  • Embodiments 8 A method for determining a risk for a protein aggregation disease (PAD) in an individual, wherein the PAD is associated with aggregation of a target protein, comprising determining the level of one or more SHD catabodies in a biological sample of the individual, wherein the individual is determined as having a risk for the PAD if the level of the one or more SHD catabodies is lower than a control SHD catabody level.
  • PAD protein aggregation disease
  • Embodiments 9 The method of embodiment 8, wherein the level of one or more SHD catabodies is the level of one or more SHD catabodies that specifically bind to the target protein.
  • Embodiments 10 The method of embodiment 8, wherein the level of one or more SHD catabodies is the level of total SHD catabodies.
  • Embodiments 11 The method of embodiment 10, wherein the level of total SHD catabodies is determined by contacting a serum sample of the individual with a substrate peptide immobilized on a solid support under conditions that allow formation of a catabody- substrate peptide complex, and determining the amount of the catabody-substrate peptide complex, wherein the substrate peptide comprises the amino acid sequence (EAR) n (SEQ ID NO: 2), wherein n is an integer between 1 and 30.
  • EAR amino acid sequence
  • Embodiments 12 The method of embodiment 11, wherein n is 3.
  • Embodiments 13 The method of embodiment 11 or 12, wherein the serum sample contains at least about 1 mg/mL Ig.
  • Embodiments 14 The method of any one of embodiments 11-13, wherein the serum sample is incubated with the substrate peptide for about 1 hour to about 16 hours.
  • Embodiments 15 The method of any one of embodiments 1 1-14, wherein the amount of the catabody-substrate peptide complex is determined using an antibody that specifically binds to total Ig.
  • Embodiments 16 The method of embodiment 15, wherein the antibody is labeled with an enzyme or a fluorescent label.
  • Embodiments 17 The method of any one of embodiments 8-16, further comprising determining the level of an auto-antibody against the target protein in a biological sample of the individual, wherein the individual is determined as having a risk for the PAD if: (i) the level of the one or more SHD catabodies is lower than a control SHD catabody level; and (ii) the level of the auto-antibody against the target protein is lower than a control auto-antibody level.
  • Embodiments 18 The method of embodiment 17, wherein the level of the autoantibody is determined by contacting a serum sample of the individual with the target protein under conditions that allow formation of an auto-antibody-target protein complex, and determining the amount of the auto-antibody-target protein complex.
  • Embodiments 19 The method of embodiment 18, wherein the level of the autoantibody is determined using an ELISA assay.
  • Embodiments 20 The method of any one of embodiments 17-19, wherein tire control auto-antibody level is the level of the auto-antibody against the target protein in a healthy individual.
  • Embodiments 21 The method of any one of embodiments 17-19, wherein the control auto-antibody level is the median level of tire auto-antibody against the target protein in a population of individuals.
  • Embodiments 22 The method of any one of embodiments 8-16, further comprising determining the level of the target protein in a biological sample of the individual, wherein the individual is determined as having a risk for the PAD if: (i) the level of the one or more SHD catabodies is lower than a control SHD catabody level; and (ii) the level of the target protein is higher than a control target protein level.
  • Embodiments 23 The method of any one of embodiments 8-22, wherein the control SHD catabody level is the level of one or more SHD catabodies in a healthy individual.
  • Embodiments 24 The method of any one of embodiments 8-22, wherein the control SHD catabody level is the median level of one or more SHD catabodies in a population of individuals.
  • Embodiments 25 The method of any one of embodiments 8-24, wherein the PAD is Alzheimer’s disease, and wherein the target protein is amyloid b (Ab).
  • Embodiments 26 The method of any one of embodiments 8-24, wherein:
  • the PAD is Parkinson’s disease, and the target protein is a-synuclein;
  • the PAD is Alzheimer’s disease or dementia, and the target protein is Tau;
  • the PAD is ATTR amyloidosis, and the target protein is transthyretin;
  • the PAD is AL amyloidosis, and the target protein is immunoglobulin light chain;
  • the PAD is frontotemporal lobar degeneration or amyotrophic lateral sclerosis, and the target protein is TDP43;
  • the PAD is Huntington’s disease, and the target protein is Huntingtin;
  • the PAD is Type II diabetes, and the target protein is LAPP; or
  • the PAD is Amyotrophic Lateral Sclerosis
  • the target protein is SOD1.
  • Embodiments 27 A method of treating or preventing a PAD in an individual, wherein the PAD is associated with aggregation of a target protein, comprising:
  • Embodiments 28 The method of embodiment 27, wherein the method is repeated at a frequency of no more than about every three months.
  • Embodiments 29 The method of embodiment 27 or 28, wherein the PAD is
  • the target protein is amyloid b (Ab)
  • the therapeutic catabody comprises a light chain variable region (VL) comprising a light chain
  • LC-CDR complementarity determining region 1 comprising the amino acid sequence of SEQ ID NO: 12, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 13, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 14, or a variant thereof comprising up to about 5 amino acid substitutions in the LC-CDRs, wherein the amino acid residue at position 1 of the VL is D, the amino acid residue at position 27A of the VL is S, and the amino acid residue at position 93 of the VL is H, and wherein the numbering is according to tire EU index of Rabat.
  • Embodiments 30 The method of embodiment 29, wherein the therapeutic catabody comprises a heavy chain variable region (VH) comprising a heavy chain complementarity determining region (HC-CDR) 1 comprising the amino acid sequence of SEQ ID NO: 9, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 10, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 11, or a variant thereof comprising up to about 5 amino acid substitutions in the HC-CDRs.
  • VH heavy chain variable region
  • HC-CDR heavy chain complementarity determining region
  • Embodiments 31 The method of embodiment 29 or 30, wherein the amino acid residue at position 26 of the VL is S, the amino acid residue at position 27D of the VL is D, E or H, and/or the amino acid residue at position 28 of the VL is D or N, and wherein the numbering is according to the EU index of Rabat.
  • Embodiments 32 The method of any one of embodiments 29-31, wherein the therapeutic catabody comprises a VH comprising an amino acid sequence having at least about 85% sequence identity to the amino acid sequence of SEQ ID NO: 4, 6, 19 or 20; and/or a VL comprising an amino acid sequence having at least about 85% sequence identity to the amino acid sequence of SEQ ID NO: 5, 7, 8, 21 or 22.
  • Embodiments 33 The method of embodiment 32, wherein the therapeutic catabody comprises: (i) a VH comprising the amino acid sequence of SEQ ID NO: 4, and a VL comprising the amino acid sequence of SEQ ID NO: 5; (ii) a VH comprising the amino acid sequence of SEQ ID NO: 6, and a VL comprising the amino acid sequence of SEQ ID NO: 7; (iii) a VH comprising the amino acid sequence of SEQ ID NO: 6, and a VL comprising the amino acid sequence of SEQ ID NO: 8; (iv) a VH comprising the amino acid sequence of SEQ ID NO: 19, and a VL comprising the amino acid sequence of SEQ ID NO: 21; (v) a VH comprising the amino acid sequence of SEQ ID NO: 20, and a VL comprising the amino acid sequence of SEQ ID NO: 21; (vi) a VH comprising the amino acid sequence of SEQ ID NO: 19, and a VL comprising the amino acid sequence of amino acid sequence
  • Embodiments 34 The method of any one of embodiments 27-33, wherein the therapeutic catabody is a full-length IgG antibody.
  • Embodiments 35 The method of embodiment 34, wherein the therapeutic catabody comprises an IgGl or IgG4 Fc region.
  • Embodiments 36 The method of any one of embodiments 27-33, wherein the therapeutic antibody is a full-length IgM antibody.
  • Embodiments 37 An isolated anti-Ab catabody comprising: a VL comprising an LC- CDR1 comprising the amino acid sequence of SEQ ID NO: 12, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 13, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 14, or a variant thereof comprising up to about 5 amino acid substitutions in the LC-CDRs, wherein the amino acid residue at position 1 of the VL is D, the amino acid residue at position 27A of the VL is S, and the amino acid residue at position 93 of the VL is H, and wherein the numbering is according to the EU index of Rabat.
  • Embodiments 38 An isolated anti-Ab catabody comprising: a VH comprising an HC- CDR1 comprising the amino acid sequence of SEQ ID NO: 9, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 10, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 11, or a variant thereof comprising up to about 5 amino acid substitutions in the HC-CDRs, wherein the amino acid residue at position 1 of the VL is D, the amino acid residue at position 27 A of the VL is S, and the amino acid residue at position 93 of the VL is H, and wherein the numbering is according to the EU index of Rabat.
  • Embodiments 39 The anti-Ab catabody of embodiment 37 or 38, wherein the anti- An catabody comprises a VH comprising an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 9, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 10, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 11; and a VL comprising an LC-CDRl comprising the amino acid sequence of SEQ ID NO: 12, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 13, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 14.
  • Embodiments 40 The anti-Ab catabody of any one of embodiments 37-39, wherein the anti-Ab catabody cleaves a substrate having the formula EAR-AMC (SEQ ID NO: 1).
  • Embodiments 41 The anti-Ab catabody of any one of embodiments 37-40, wherein the amino acid residue at position 26 of the VL is S, the amino acid residue at position 27D of the VL is D, E or H, and/or the amino acid residue at position 28 of the VL is D or N, and wherein the numbering is according to the EU index of Rabat.
  • Embodiments 42 The anti-Ab catabody of any one of embodiments 37-41, wherein the anti-Ab catabody comprises a VH comprising an ammo acid sequence having at least about 85% sequence identity to the amino acid sequence of SEQ ID NO: 4, 6, 19 or 20; and/or a VL comprising an amino acid sequence having at least about 85% sequence identity to the amino acid sequence of SEQ ID NO: 5, 7, 8, 21 or 22.
  • Embodiments 43 The anti-Ab catabody of embodiment 42, wherein the anti-Ab catabody comprises: (i) a VH comprising the amino acid sequence of SEQ P) NO: 4, and a VL comprising the amino acid sequence of SEQ ID NO: 5; (ii) a VH comprising the amino acid sequence of SEQ ID NO: 6, and a VL comprising the amino acid sequence of SEQ ID NO: 7; (iii) a VH comprising the amino acid sequence of SEQ ID NO: 6, and a VL comprising the amino acid sequence of SEQ ID NO: 8; (iv) a VH comprising the amino acid sequence of SEQ ID NO: 19, and a VL comprising the amino acid sequence of SEQ ID NO: 21 ; (v) a VH comprising the amino acid sequence of SEQ ID NO: 20, and a VL comprising the amino acid sequence of SEQ ID NO: 21; (vi) a VH comprising the amino acid sequence of SEQ ID NO: 19, and
  • Embodiments 44 The anti-Ab catabody of any one of embodiments 37-43, wherein the anti-Ab catabody is a foll-length IgG antibody.
  • Embodiments 45 The anti-Ab catabody of embodiment 44, wherein the anti-Ab catabody comprises an IgGl or IgG4 Fc region.
  • Embodiments 46 The anti-Ab catabody of any one of embodiments 37-43, wherein the anti-Ab catabody is a full-length IgM antibody.
  • Embodiments 47 A method of treating or preventing Alzheimer’s disease in an individual, comprising administering to the individual an effective amount of the anti-Ab catabody of any one of embodiments 37-46.
  • Embodiments 48 A kit for treating or preventing Alzheimer’s disease in an individual, comprising:
  • a substrate peptide comprises the amino acid sequence (EAR) n (SEQ ID NO: 2), wherein n is an integer between 1 and 30;
  • Embodiments 49 The kit of embodiment 48, further comprising a solid support.
  • Embodiments 50 The kit of embodiment 48 or 49, further comprising a therapeutic catabody that specifically binds to Ab.
  • SHD catabodies i.e. , catabodies having an“SHD” motif
  • PAD protein aggregation diseases
  • Serum SHD catabody levels in young adults (20 to 29 years old; HS2, HS6, HS7 and HS8) and old adults (60 to 69 years old; HS1, HS3, HS4 and HS5) were measured using two immunoassays.
  • EAR-AMC binding assay an ELISA plate was coated with an EAR peptide (SEQ ID NO: 1) conjugated to 7-amino-4-methylcoumarin (AMC) (“EAR-AMC,” 100X, Bachem Americas, Inc. Cat. No. 1-1575.0050).
  • AMC 7-amino-4-methylcoumarin
  • EARS binding assay an ELISA plate was coated with an (EAR).3 peptide (SEQ ID NO: 3).
  • each ELISA plate was coated with the corresponding peptide at a final concentration of about 5 mg/mL in a coating buffer (0.2M sodium carbonate-bicarbonate buffer, pH 9.4), 100 pL/well at 4°C overnight.
  • the plates were washed three times with a washing buffer (PBST: 0.1% Tween-20 in phosphate buffer saline“PBS”).
  • PBST 0.1% Tween-20 in phosphate buffer saline“PBS”.
  • the plates were then blocked with a blocking buffer (1% BSA in PBS) for 1 hour at room temperature, and w'ashed three times with the washing buffer.
  • To each well of the plate was added 100 pL of the corresponding serum sample (25mg/mL or lOOmg/mL), and PBS was used as negative control.
  • HRP substrate Amplex Red and H2O2
  • a developing buffer 3.6 mM NaaHPO*, 1.4 mM NaHzPCU, at pH7.2
  • FIG. 1A shows the results of the EAR-AMC binding assay
  • FIG. IB shows the results of the EAR3 binding assay.
  • the human serum pool (HS pool) and 3 out of 4 serum samples from the young adult group (20-29 years old) have higher semm SHD catabody levels compared to 3 out of 4 serum samples from the old adult group (60-69 years old).
  • This result suggests that reduction of serum SHD catabodies might be an age-related event and serum level of SHD catabodies may serve as a biomarker to predict protein aggregation diseases.
  • catabody levels and Ab-specific auto-antibody levels in serum samples from healthy individuals (HS1-8) and Alzheimer’s disease patients (ALZ1-5) were determined using two immunoassays.
  • EAR3 binding assay an ELISA plate was coated with an (EAR)3 peptide (SEQ ID NO: 3) at a final concentration of 5 mg/mL.
  • Ab binding assay an ELISA plate was coated with a biotinylated amyloid (1-42) peptide at a final concentration of 1 mg/mL.
  • each ELISA plate was coated with the corresponding peptide in a coating buffer (0.2M sodium carbonate-bicarbonate buffer, pH 9.4), 100 pL/well at 4°C overnight. The plates were washed three times with a washing buffer (PBST). The plates were then blocked with a blocking buffer (10% Fetal Bovine Seram) for 1 hour at room temperature, and washed three times with the washing buffer. To each well of the plate was added 100 pL of the corresponding serum sample (100 mg/mL), and PBS was used as negative control. After incubation for 1 hour, the plates were then washed three times with the washing buffer, and blocked with the blocking buffer for 1 hour at room temperature.
  • a coating buffer 0.2M sodium carbonate-bicarbonate buffer, pH 9.4
  • PBST washing buffer
  • a blocking buffer 10% Fetal Bovine Seram
  • FIG. 2 shows the results of the (EAR)a and Ab binding assays. Compared to the healthy individuals, 4 out of the 5 AD patients had significant reduction in both the catabody and Ab-specific auto-antibody serum levels. This result suggests that co-reduction of serum SHD catabody levels and Ab-specific auto-antibody levels can serve as a biomarker for AD diagnosis.
  • This example describes the design and characterization of anti-Ab catabodies based on 3D6, a non-catalytic antibody that specifically binds Ab.
  • Peripheral Ab hydrolysis may induce depletion of the brain Ab storage without IgM passage across the BBB.
  • IgMs mediate the innate immune response, usually have lower affinity to target antigens, and are more difficult to manufacture.
  • SHD catabodies are known to have an SHD motif is the light chain variable region (VL).
  • VL light chain variable region
  • a 3D6-D antibody was engineered by replacing the Y residue at position 1 of the VL of 3D6 with a D, and expressed recombinantly and purified.
  • FIG. 4 shows reducing and nonreducing gel electrophoresis of 3D6-D and 3D6 (i.e., 3D6-Y) antibodies, and humanized versions thereof. Sequences of the 3D6-D catabody are shown in Table 2.
  • the catalytic activity of the 3D6-D antibody was assessed in an EAR-AMC catalytic function assay. Briefly, each well of an ELISA plate was coated with an (100X, Bachem Americas, Inc. Cat. No. 1-1575.0050) at a 1: 100 dilution. 3D6-D (400 ng/mL), 3D6-Y (400ng/mL), trypsin (0.25% trypsin-EDTA at 1:10,000 dilution) was each mixed with an enzymatic assay buffer (50 mM Tris-HCl, pH7.7, 0.1M Glycine, 0.025% Tween-20) and EAR-AMC (100 mM) in PBS buffer.
  • an enzymatic assay buffer 50 mM Tris-HCl, pH7.7, 0.1M Glycine, 0.025% Tween-20
  • EAR-AMC 100 mM
  • EAR-AMC is a substrate of SHD catabodies, which cleave EAR-AMC at the covalent bond between Arg and AMC, thereby releasing AMC.
  • Binding of 3D6-D, 3D6, or isotype control (IgGl) to (EAR)3 and Ab were determined using the EAR3 binding assay and Ab binding assay respectively as described in Example 1. Each antibody was added to the well of the ELISA plate at a concentration of 100 mg/mL. As shown in FIG. 6, 3D6-D has comparable (EAR)i and Ab-binding activity as the 3D6 antibody, which suggests that 3D6-D is a high affinity IgGl catabody for Ab.
  • humanized catabodies were produced by grafting CDRs of 3D6-D into human antibody framework sequences. Back-mutations in the human antibody framework sequences to original mouse sequences were further introduced to maintain the affinity of the humanized antibody and to facilitate further development of the antibody. Additionally, the SHD motif and other residues that may support SHD motif catalytic functions (e.g., residues marked in FIG. 3) and/or maintain the conformational structure of the SHD motif were back-mutated to the original mouse sequences. Exemplary humanized 3D6-D antibodies sequences are shown in Table 2.
  • Binding affinities of humanized 3D6-D catabodies to Ab were determined using an Ab binding assay. Briefly, an ELISA plate was coated with an Ab (1-42) peptide at 1 mg/mL in a coating buffer (0.2M sodium carbonate-bicarbonate buffer, pH 9.4), 100 pL/well at 4°C overnight. The Ab peptide was removed and the plate was washed three times with 250 pL/well of a washing buffer (PBST). The plate was then blocked with 200 pL/well of a blocking buffer ( 1% BSA in PBST) for 1 hour at room temperature, and washed two times with 250 pL/well of the washing buffer.
  • a coating buffer 0.2M sodium carbonate-bicarbonate buffer, pH 9.4
  • a human scFv/Fab phage library with human germline VHS and the humanized VL sequences of 3D6-D is panned against Ab to select for humanized anti-Ab catalytic scFvs or Fabs.
  • the HC-CDR sequences in the phage library are randomized.
  • the scFvs or Fabs are selected based on high specificity for Ab.
  • the catalytic activity of the selected scFvs and Fabs is assessed using the EAR-AMC catalytic function assay described in Example 2.
  • the selected anti-Ab scFvs and Fabs are used to make full-length IgG (e.g., IgGl or IgG4) catabodies.
  • This experiment was carried out to detect anti-Ab autoantibody levels and SHD catabody (recognizing (EAR)? peptide) levels in the serum of Alzheimer’s disease (AD) patients versus that of healthy individuals.
  • AD serum samples used in this assay included 25 new AD patient serum samples and 5 old AD patient serum samples from Example 1.
  • 8 healthy donor serum (HS) samples came from young adults (20 to 29 years old; HS2, HS6, HS7 and HS8) and old adults (60 to 69 years old; HS1, HS3, HS4 and HS5), as used in Example 1.
  • One pooled healthy donor serum sample served as positive control (NHS; Innovative Research).
  • PBS served as negative control.
  • Total 40 test samples were centrifuged at 16000 g for 10 min, and supernatant was collected for ELISA assays. Each sample was tested in duplicates.
  • an ELISA plate was coated with an (EAR)? peptide (SEQ ID NO: 3).
  • (EAR)? stock (5 mg/mL) was diluted 100 folds with coating buffer (0.2M sodium carbonate-bicarbonate buffer, pH 9.4), with a final concentration of 50 mg/mL, coated 100 pL/well, 4°C overnight.
  • coating buffer 0.2M sodium carbonate-bicarbonate buffer, pH 9.4
  • biotinylated amyloid (1-42) peptide at a final concentration of 2 mg/mL (1 mg/mL stock was diluted 500 folds) in coating buffer (0.2M sodium carbonate-bicarbonate buffer, pH 9.4), 100 pL/wcll, 4°C overnight.
  • the plates were washed three times with 200 pL washing buffer (PBST: 0.1% Tween-20 in phosphate buffer saline“PBS”) each time.
  • the plates were then blocked with a blocking buffer (1% BSA in PBST) fir 2 hours at room temperature, and washed three times with 200 pL washing buffer (PBST) each time.
  • PBST phosphate buffer saline
  • To each well of the plate was added 100 pL of the corresponding test sample at 1:100 dilution with PBS. All samples were tested in duplicates for each peptide target. See FIG. 9A for loading design. After incubation at 4°C overnight, the plates were then washed three times with 200 pL washing buffer (PBST) each time.
  • SEQ ID NO: 3 substrate peptide amino acid sequence
  • SEQ ID NO: 4 anti-Ab catabody 3D6 VH amino acid sequence; CDRs are underlined
  • SEQ ID NO: 9 anti-Ab catabody HC-CDR1 amino acid sequence
  • SEQ ID NO: 12 anti-Ab catabody LC-CDR1 amino acid sequence
  • SEQ ID NO: 13 anti-Ab catabody LC-CDR2 amino acid sequaice
  • SEQ ID NO: 14 anti-Ab catabody LC-CDR3 amino acid sequence
  • SEQ ID NO: 16 (IgG4 heavy chain constant region amino acid sequence)
  • HNHYTQKSLSLSLGK SEQ ID NO: 17 (light chain kappa constant region amino add sequence)
  • SEQ ID NO: 18 (light chain lambda constant region amino acid sequence)
  • SEQ ID NO: 24 bapineuzumab VH amino acid sequence; CDRs are underlined
  • SEQ ID NO: 25 bapineuzumab VL amino acid sequence; CDRs are underlined

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Abstract

La présente invention concerne des méthodes, des compositions et des kits pour déterminer des niveaux d'anticorps catalytiques SHD dans un échantillon biologique, et pour traiter ou prévenir une maladie d'agrégation protéique (PAD) chez un individu. L'invention concerne également des anticorps catalytiques qui reconnaissant spécifiquement des peptides β-amyloïdes (Αβ) et leurs méthodes d'utilisation.
PCT/US2019/046903 2018-08-17 2019-08-16 Anticorps catalytiques et leurs méthodes d'utilisation WO2020037258A1 (fr)

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